prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/7917/1/a zaheer thesis 20... · thesis...

Bacterial Diversity in the Root Nodules and

Rhizosphere of Chickpea (Cicer arietinum L.)

Ahmad Zaheer

2017

Department of Biotechnology

Pakistan Institute of Engineering & Applied Sciences

Nilore-45650 Islamabad, Pakistan

ii

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Thesis Submission Approval

This is to certify that the work contained in this thesis entitled Bacterial Diversity in

the Root Nodules and Rhizosphere of Chickpea (Cicer arietinum L.), was carried

out by Ahmad Zaheer, and in my opinion, it is fully adequate, in scope and quality,

for the degree of Ph.D. Furthermore, it is hereby approved for submission for review

and thesis defense.

Supervisor: __________________________

Name: Dr. Muhammad Sajjad Mirza

Date: 13 July, 2017

Place: NIBGE, Faisalabad.

Co-Supervisor: _______________________

Name: Dr. Kauser A. Malik HI, SI, TI

Date: 13 July, 2017

Place: F.C. College (A Chartered

University), Lahore.

Head, Department of Biotechnology: _________________________

Name: Dr. Shahid Mansoor SI

Date: 13 July, 2017

Place: NIBGE, Faisalabad.

Bacterial Diversity in the Root Nodules and

Rhizosphere of Chickpea (Cicer arietinum L.)

Ahmad Zaheer

Submitted in partial fulfillment of the requirements

for the degree of Ph.D.

2017

Department of Biotechnology

Pakistan Institute of Engineering and Applied Sciences

Nilore-45650 Islamabad, Pakistan

ii

Dedications

To

My Father and Mother

iii

Acknowledgements

Nothing is deserving of worship except “ALMIGHTY ALLAH”, all praises for Him,

Who is the entire source of all knowledge and wisdom endowed to mankind. He guides

the way and gives me courage to complete this work. I offer my humblest gratitude

from deep sense of heart to the Holy Prophet, MUHAMMAD (صلى هللا عليه وسلم) Who

is, forever source of guidance and knowledge for humanity.

First and foremost, I offer my sincerest gratitude to my supervisor, Dr.

Muhammad Sajjad Mirza, Deputy Chief Scientist, National Institute for

Biotechnology and Genetic Engineering (NIBGE), for his constant guidance, kind

supervision and valuable help at every step of my PhD study. I am thankful to my co-

supervisor Professor Dr. Kauser A. Malik (H.I., S.I., T.I.), Distinguished National

Professor, Forman Christian College University, Lahore, for his kind and affectionate

behavior, personal interest and valuable guidance.

I would also like to appreciate and acknowledge the help and support of Dr.

Shahid Mansoor (S.I.), Director NIBGE, for providing me the opportunity to carry out

research work at NIBGE.

I am also grateful to my foreign supervisors Professor Dr. Xavier Perret and

Dr. Maged M. Saad at Microbiology Unit, Department of Botany and Plant Biology,

University of Geneva, Switzerland for their technical support and valuable contribution

during my visit to the host lab. I am sincerely thankful to my UNIGE lab fellows Dr.

Antoine Huyghe, Romain Fossou, Laura Piazza and Natalia Giot for their help and

support. I am thankful to foreign collaborators Dr. Joan E. Mclean and Dr. Babur S.

Mirza at Utah Water Research Laboratory, Utah State University, Logan, Utah, USA

for their financial and technical support in pyrosequencing and data analysis.

Help and cooperation from Dr. Tariq Mahmud Shah, Deputy Chief Scientist,

Head PBG, Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad, Dr.

Khalid Hussain, Director, Arid Zone Research Institute, Bhakkar and Malik Mushtaq

iv

Ahmad, Assistant Botanist, Incharge Pulses Research Sub-Station, Kallurkot by

providing chickpea seeds and land to conduct multi-locational field experiments is

thankfully acknowledged.

Heartiest thanks to Dr. Ghulam Rasul, Dr. Fathia Mubin, Dr. Sumera

Yasmin and Dr. Asma Imran, who helped me throughout my PhD study. I am also

thankful to my lab collogues Dr. Muther Mansoor Qaisrani, Dr. Muhammad Tahir,

Dr. Muhammad Arshad, Ms. Aamna Basheer, Ms. Khadija Ayyaz and Ms. Sughra

Hakim for their support during the whole period of my research work. I am also

thankful to lab technicians Muhammad Ahmad Dogar, Zahid Iqbal, Zakir Hussain,

Ghulam Abas and Imran ul Haq for the kind help in conducting lab and field

experiments.

Thank you to my family and friends who, through their love, support and

encouragement, have helped to make my dreams a reality. I do not have words to

express my heartiest thanks, gratitude and profound admiration to my affectionate

parents, who are the source of encouragement for me in fact this work became possible

only because of their love, moral support and prayers for my success. Whatever, I am

today is because of their love and prayers.

I wish to acknowledge financial support from the Higher Education

Commission (HEC) of Pakistan under the program PhD Fellowship Batch VI and

International Research Support Initiative Programme (IRSIP) and Pakistan Science

Foundation (PSF project No. 315).

Ahmad Zaheer

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Declaration of Originality

I hereby declare that the work accomplished in this thesis is the result of my own

research carried out in Soil & Environmental Biotechnology Division (NIBGE). This

thesis has not been published previously nor does it contain any material from the

published resources that can be considered as the violation of international copyright

law. Furthermore, I also declare that I am aware of the terms ‘copyright’ and

‘plagiarism’, and if any copyright violation was found out in this work I will be held

responsible of the consequences of any such violation.

__________________

(Ahmad Zaheer)

13 July, 2017

NIBGE, Faisalabad.

vi

Copyrights Statement

The entire contents of this thesis entitled Bacterial Diversity in the Root Nodules and

Rhizosphere of Chickpea (Cicer arietinum L.) by Ahmad Zaheer are an intellectual

property of Pakistan Institute of Engineering & Applied Sciences (PIEAS). No portion

of the thesis should be reproduced without obtaining explicit permission from PIEAS.

vii

Table of Contents

Dedications .................................................................................................................... ii

Acknowledgements ...................................................................................................... iii

Declaration of Originality .............................................................................................. v

Copyrights Statement .................................................................................................... vi

Table of Contents ......................................................................................................... vii

List of Figures ................................................................................................................ x

List of Tables ............................................................................................................... xii

Abstract ....................................................................................................................... xiv

List of Publications and Patents ................................................................................... xv

List of Abbreviations and Symbols............................................................................. xvi

1 Introduction ................................................................................................................. 1

1.1 Chickpea Crop ..................................................................................................... 1

1.2 The Chickpea-Mesorhizobium Symbiosis ........................................................... 2

1.3 Bacterial Diversity Associated with the Root Nodules ....................................... 2

1.4 The Rhizospheric Soil ......................................................................................... 3

1.4.1 Identification of Bacterial Isolates from Rhizospheric Soil ................... 4

1.5 Mechanisms used by Bacteria for Plant Growth Promotion ............................... 4

1.5.1 Biological Nitrogen Fixation and Nodulation........................................ 5

1.5.2 Phytohormone Production ..................................................................... 6

1.5.3 Phosphate Solubilization ........................................................................ 7

1.6 Application of Bacterial Inoculation for Plant Growth Promotion ..................... 8

1.7 Metagenomics ...................................................................................................... 8

1.8 Objectives of the Present Study ......................................................................... 10

2 Materials and Methods .............................................................................................. 11

2.1 Sample Collection.............................................................................................. 11

2.2 Soil Analysis ...................................................................................................... 11

2.3 Bacterial Isolation .............................................................................................. 12

2.3.1 Isolation of Endophytic Bacteria from Nodules of Chickpea .............. 12

2.3.2 Isolation of PGPR from Rhizospheric Soil of Chickpea ..................... 13

2.4 Molecular Characterization and Identification of Bacterial Isolates ................. 14

2.4.1 Extraction of Genomic DNA from Pure Cultures................................ 14

viii

2.4.2 Identification by nifH and 16S rRNA Gene Amplification ................. 14

2.4.3 Phylogenetic Analysis of the Bacterial Isolates ................................... 14

2.5 Preservation of Bacteria ..................................................................................... 15

2.6 Characterization of Bacterial Isolates ................................................................ 15

2.6.1 Confirmation of Nodulation Ability by Endophytic Bacterial Isolates

on Chickpea ......................................................................................... 15

2.6.2 Indole-3-Acetic Acid (IAA) Production by Isolates ............................ 15

2.6.3 Phosphate Solubilization by the Bacterial Isolates in Pure Culture ..... 16

2.6.4 Organic Acid Production by the Bacterial Isolates in Pure Culture .... 17

2.7 Effect of Bacterial Inoculations on Chickpea .................................................... 17

2.7.1 Earthen Pot Experiments to Study the Effect of Bacterial Inoculation

on Chickpea ......................................................................................... 17

2.7.2 Field Trials to Study the Effect of Bacterial Inoculation on Chickpea 17

2.8 Bacterial Diversity in Rhizospheric Soil and Root Nodules Studied by Culture-

Independent DNA-Based (16S rRNA and nifH Genes Sequences) Method ..... 18

2.9 Statistical Analysis ............................................................................................ 19

2.10 Nucleotide Sequence Accession Numbers ........................................................ 19

3 Results ....................................................................................................................... 21

3.1 Isolation and Identification of Bacterial Isolates ............................................... 21

3.1.1 Isolation and Identification of Bacteria from Rhizospheric Soil and

Root Nodules of Chickpea ................................................................... 21

3.1.2 Identification of Bacterial Isolates ....................................................... 26

3.1.3 Amplification of nifH Gene from Bacterial Isolates ............................ 38

3.2 Characterization of the Bacterial Isolates .......................................................... 39

3.2.1 Confirmation of Nodulation Ability of Endophytic Bacterial Isolates 39

3.2.2 Indole-3-Acetic Acid (IAA) Production by the Bacterial Isolates ....... 42

3.2.3 Phosphate Solubilization by the Bacterial Isolates .............................. 43

3.2.4 Organic Acid Production ..................................................................... 47

3.3 Effect of Bacterial Inoculation on Chickpea ..................................................... 49

3.3.1 Effect of Bacterial Inocula on Chickpea Plants Grown in Earthen Pots

(Year 2012-13) ..................................................................................... 49

3.3.2 Plant Growth Promoting Effect of Serratia spp. on Chickpea Grown in

Field (Year 2013-14)............................................................................ 52

3.3.3 Plant Growth Promoting Effect of Bacterial Inocula on Chickpea

Grown in Earthen Pots (Year 2013-14) ............................................... 52


Grown in Field (Year 2014-15) ........................................................... 60

ix


Grown at Different Locations (Year 2015-16) .................................... 61

3.4 Bacterial Diversity by Culture-Independent Molecular Approach .................... 71

3.4.1 Extraction of DNA and PCR Amplification of 16S rRNA and nifH

Genes from the Root Nodules and Rhizospheric Soil of Chickpea ..... 71

3.4.2 Bacterial Diversity in the Root Nodules Revealed by Sequence

Analysis of nifH gene Amplified from Nodule DNA .......................... 71

3.4.3 Bacterial Diversity Revealed by Sequence Analysis of nifH Gene

Amplified from Rhizospheric Soil DNA ............................................. 73


Analysis of 16S rRNA Gene Amplified from Nodule DNA ............... 75

3.4.5 Bacterial Diversity Revealed by Sequence Analysis of 16S rRNA Gene

Amplified from Rhizospheric Soil DNA ............................................. 81

4 Discussion ................................................................................................................. 86

Appendices ................................................................................................................... 95

References .................................................................................................................. 101

x

List of Figures

Figure 1-1 Seeds and flowers of Desi, Kabuli-type chickpea ................................. 1

Figure 2-1 Map of Pakistan showing different sampling locations ....................... 12

Figure 3-1 Sample collection from different chickpea growing area. ................... 21

Figure 3-2 Colony morphology of the isolates on LB and YMA media. .............. 22

Figure 3-3 Genomic DNA extracted from isolates. ............................................... 27

Figure 3-4 PCR-amplification of 16S rRNA gene from bacterial isolates. ........... 29

Figure 3-5 16S rRNA sequence-based phylogenetic tree of α-Proteobacteria ..... 30

Figure 3-6 16S rRNA sequence-based phylogenetic tree of β-Proteobacteria ..... 31

Figure 3-7 16S rRNA sequence-based phylogenetic tree of γ-Proteobacteria ..... 32

Figure 3-8 16S rRNA sequence-based phylogenetic tree of Actinobacteria ......... 33

Figure 3-9 16S rRNA sequence-based phylogenetic tree of Firmibacteria .......... 34

Figure 3-10 PCR amplification of partial nifH gene from Mesorhizobium . ........... 38

Figure 3-11 nifH sequence-based phylogenetic tree of Mesorhizobium ................. 39

Figure 3-12 Effect of Mesorhizobium on growth and nodulation of chickpea. ....... 40

Figure 3-13 Nodulation of chickpea by pure cultures of Mesorhizobium spp. ....... 40

Figure 3-14 Qualitative test showing IAA production by bacterial isolates. .......... 43

Figure 3-15 IAA production by selected strains at different incubation temp. ....... 44

Figure 3-16 Plate assay for P solubilizing activity of NFY8 and 5D. ..................... 44

Figure 3-17 P solubilization by selected strains at different incubation temp. ........ 45

Figure 3-18 Organic acid production by bacterial strains. ...................................... 48

Figure 3-19 Effect of bacterial strains on growth of chickpea plants grown in

earthen pots. Strain used: Serratia sp. 5D. (Year 2012-13) ................. 49

Figure 3-20 Effect of Serratia sp. on growth of chickpea plants grown in field at

two different localities. (Year 2013-14)............................................... 53

Figure 3-21 Effect of inoculation (Serratia spp.) on grain yield of chickpea ......... 54

Figure 3-22 Effect of inoculation (Serratia spp.) on straw weight of chickpea ...... 55

Figure 3-23 Effect of bacterial inocula on growth of chickpea plants grown in

Earthen pots.-------------------------------------------------------------------56

Figure 3-24 Effect of bacterial inocula on growth of chickpea plants grown in field

at NIBGE, Faisalabad. (Year 2014-15)................................................ 62

Figure 3-25 Effect of bacterial inoculation on chickpea grown in field. ................. 63

xi

Figure 3-26 Effect of bacterial strains on growth of chickpea plants grown in field

at different localities. (Year 2015-16) .................................................. 63



Figure 3-29 Agarose gels showing DNA extracted from root nodules and PCR

amplification of 16S rRNA and nifH genes ......................................... 71

Figure 3-30 Relative abundance of major bacterial classes detected by 16S rRNA

gene sequence analysis in the root nodules of chickpea grown at

different localities ------------------------------------------------------------77

Figure 3-31 Relative abundances of the major bacterial genera detected by 16S

rRNA gene sequence analysis in nodules of chickpea ........................ 78

Figure 3-32 Molecular phylogenetic analysis of the 16S rRNA sequences retrieved

from root nodules of chickpea. ............................................................ 79

Figure 3-33 Non-metric multi-dimensional scaling representation of the

geochemical characteristics and relative abundance of the Serratia

sequences in the root nodules of chickpea ........................................... 79

Figure 3-34 16S rRNA sequence-based phylogenetic tree of Serratia strains

isolated from root nodule of chickpea constructed by maximum

likelihood method. .............................................................................. 80

Figure 3-35 Relative abundance of major bacterial phyla detected by 16S rRNA

gene sequence analysis from rhizospheric soil of chickpea grown at

different localities. ............................................................................... 82

xii

List of Tables

Table 3.1 Physio-chemical characteristics of soil samples collected from different

localities ............................................................................................... 23

Table 3.2 Morphological characteristics of the bacterial isolates obtained from

rhizospheric soil and root nodules of chickpea .................................... 24

Table 3.3 Identification of the bacterial isolates obtained from rhizospheric soil

and root nodules of chickpea on the basis of 16S rRNA gene ............. 35

Table 3.4 Nodulation of Desi-type chickpea by pure cultures of endophytes ..... 41

Table 3.5 Nodulation of Kabuli-type chickpea by pure cultures of endophytes.. 42

Table 3.6 Production of IAA (µg/mL) and Phosphate solubilization (µg/mL) by

bacterial strains in the growth medium. ............................................... 45

Table 3.7 Production of IAA by bacterial strains at different temperatures ........ 47

Table 3.8 Phosphate solubilization by bacterial strains at different temp ........... 47

Table 3.9 Organic acid production by strains in Pikovskaya growth medium .... 48

Table 3.10 Effect of bacterial inocula on number of nodules and dry weight of

nodules of chickpea plant grown in earthen pots. (Year 2012-13) ...... 50

Table 3.11 Effect of bacterial isolates on grain and straw yield (g/plant) of chickpea

grown in earthen pots. (Year 2012-13) ................................................ 51

Table 3.12 Characteristics of soil samples collected at the time of sowing ........... 56

Table 3.13 Effect of bacterial inoculation on grain yield of chickpea . -------------57

Table 3.14 Effect of bacterial inoculation on straw yield of chickpea --------------58

Table 3.15 Effect of bacterial isolates as single-strain inocula and as co-inoculants

on chickpea grown in earthen pots. (Year 2013-14) ............................ 59

Table 3.16 Effect of isolates as single strain inocula and co-inoculation on grain

and straw yield of chickpea grown in earthen pots. (Year 2013-14) ... 60

Table 3.17 Effect of bacterial inoculation on number of nodules and dry weight of

nodules of chickpea grown at experimental field. (Year 2014-15) ..... 65

Table 3.18 Effect of bacterial inoculation on grain and straw yield of chickpea

grown at experimental field. (Year 2014-15) ...................................... 66

Table 3.19 Characteristics of field soil from different localities ........................... 67

Table 3.20 Effect of bacterial inoculation on nodulation of chickpea grown in

experimental fields at different locations. (Year 2015-16) .................. 68

Table 3.21 Effect of inoculation on dry weight of nodules per plant of chickpea

grown in experimental fields at different locations. (Year 2015-16) ... 68

xiii

Table 3.22 Effect of bacterial inoculation on grain yield (kg/ha) of chickpea grown

in experimental fields at different locations. (Year 2015-16) .............. 69

Table 3.23 Effect of bacterial inoculation on straw yield (kg/ha) of chickpea grown

in experimental fields at different locations. (Year 2015-16) .............. 70

Table 3.24 Dominant bacterial genera detected by nifH gene sequences amplified

from nodules of chickpea grown at different localities ....................... 72

Table 3.25 Mesorhizobial sequences detected by nifH gene amplification from

nodules of chickpea grown at different localities ................................ 73

Table 3.26 Bacterial genera detected by nifH gene amplification from rhizospheric

soil of chickpea grown at different localities ....................................... 74

Table 3.27 Mesorhizobial sequences detected by nifH gene amplified from

rhizospheric soil of chickpea grown at different localities .................. 75

Table 3.28 Relative abundance of bacterial phyla detected by 16S rRNA gene

sequence analysis in the root nodules of chickpea grown at different

localities. .............................................................................................. 80

Table 3.29 Relative abundance of bacterial classes detected by 16S rRNA gene

sequence analysis from nodules of chickpea grown in different

localities. .............................................................................................. 81


sequence analysis in the rhizospheric soil of chickpea grown at different

localities. .............................................................................................. 83

Table 3.31 Relative abundance of major bacterial classes detected by 16S rRNA

gene sequence analysis from rhizospheric soil of chickpea grown at

different localities. ............................................................................... 83

xiv

Abstract

The main objective of the present study was to study bacterial diversity in the root

nodules and rhizosphere of chickpea varieties growing in different regions of Pakistan

by cultivation on growth media as well as by using culture-independent DNA-based

techniques. A total of 60 isolates, including symbiotic (10 isolates) as well as free-living

bacteria (50 isolates), were purified from “Desi-type” and “Kabuli-type” chickpea

varieties collected from 5 different localities. In pure culture, maximum IAA

production was recorded in Kocuria sp. RTL99 (37.77 µg/mL) and maximum

phosphate solubilization was recorded in Serratia sp. 5D (119.94 µg/mL). Among the

bacterial inocula tested in pot and field experiments, co-inoculation of Mesorhizobium

sp. NTY7 and Ensifer sp. NFY8 was found to be the most effective treatment at all

localities and on both varieties of chickpea. To investigate bacterial diversity by culture-

independent DNA-based technique, DNA extracted from root nodules and rhizospheric

soil was used for pyrosequencing of 16S rRNA and nifH genes. 16S rRNA sequences

originating from the nodules revealed occurrence of 10 bacterial phyla. At genus level,

16S rRNA sequences of 111 genera (70.78 % of the total sequences) of culturable

bacteria were retrieved from nodule DNA along with 29.22 % sequences of

“uncultured” bacteria. In the nodules, a significant fraction i.e., 52.77 % of 16S rRNA

sequences and 88.83 % of the nifH sequences among the total sequences retrieved from

all sites belonged to genus Mesorhizobium. The 16S rRNA sequences originating from

the rhizospheric soil revealed enormous diversity of 22 bacterial phyla. At genus level,

16S rRNA sequences of 313 genera (29.72 % of the total sequences) of culturable

bacteria were retrieved from rhizospheric soil DNA along with 70.28 % sequences of

“uncultured” bacteria. Mesorhizobial 16S rRNA and nifH sequences retrieved from

rhizospheric soil comprised 0.265 % and 16.68 % of the total recovered sequences,

respectively. In the present study, sequences related to well-known plant growth

promoting rhizobacteria Serratia spp. were frequently detected, which lead to targeted

isolation of two Serratia strains from the nodules. Both the isolates showed growth

improvement of chickpea when used as inoculants for chickpea grown at different

localities.

xv

List of Publications and Patents

Journal Publications

• M. Tahir, M. S. Mirza, A. Zaheer, M. R. Dimitrov, H. Smidt, and S. Hameed,

"Isolation and identification of phosphate solubilizer Azospirillum, Bacillus and

Enterobacter strains by 16S rRNA sequence analysis and their effect on growth

of wheat (Triticum aestivum L.)," Australian Journal of Crop Science, vol. 7,

pp. 1284-1292, 2013.

• M. M. Qaisrani, M. S. Mirza, A. Zaheer, and K. A. Malik, "Isolation and

identification by 16S rRNA sequence analysis of Achromobacter, Azospirillum

and Rhodococcus strains from the rhizosphere of maize and screening for the

beneficial effect on plant growth," Pakistan Journal of Agricultural Sciences,

vol. 51, pp. 91-99, 2014.

• A. Basheer, A. Zaheer, M. M. Qaisrani, G. Rasul, S. Yasmin, and M. S. Mirza.,

"Development of DNA markers for detection of inoculated plant growth

promoting bacteria in the rhizosphere of wheat (Triticum aestivum L.),"

Pakistan Journal of Agricultural Sciences, vol. 53, pp. 135-142, 2016.

• K. Ayyaz, A. Zaheer, G. Rasul, and M. S. Mirza, "Isolation and identification

by 16S rRNA sequence analysis of plant growth-promoting azospirilla from the

rhizosphere of wheat," Brazilian Journal of Microbiology, vol. 47, pp. 542-550,

2016.

• A. Zaheer, B. S. Mirza, J. E. McLean, S. Yasmin, T. M. Shah, K. A. Malik, et

al., "Association of plant growth-promoting Serratia spp. with the root nodules

of chickpea," Research in Microbiology, vol. 167, pp. 510-520, 2016.

Manuals

• M. S. Mirza, S. Hameed, G. Rasul, F. Mubeen, A. Imran, A. Zaheer and M. M.

Qaisrani, “Bacterial Identification and Metagenomics,” Course manual, 2013.

ISBN: 978-969-8189-20-4

xvi

List of Abbreviations and Symbols

ºC Degree Centigrade

ºE Degree East

ºN Degree North

µL Micro Litre

µm Micro Meter

µg Micro Gram

µM Micro Molar

10X 10 times

ANOVA Analysis of Variance

AARI Ayub Agricultural Research Institute

AZRI Arid Zone Research Institute

BNF Biological Nitrogen Fixation

bp Base Pair

cfu Colony Forming Units

cm Centimeter

CTAB Cetyl Trimethylammonium Bromide

DNA Deoxyribonucleic Acid

dNTP Deoxynucleotide Triphosphates

EC Electrical Conductivity

g Gram

h Hours

HPLC High Performance Liquid Chromatography

IAA Indole-3-Acitic Acid

K Potassium

kb Kilo Base

kg Kilogram

LB Luria-Bertani

LSD Least Significant Difference

xvii

m Meter

mg Milli Gram

min Minutes

mL Milli Liter

Mo Molybdenum

N Nitrogen

NCBI National Center for Biotechnology Information

NFM Nitrogen Free Malate

NIAB Nuclear Institute for Agriculture and Biology

NIBGE National Institute for Biotechnology and Genetic Engineering

NIFA Nuclear Institute for Food and Agriculture

OTU Operational Taxonomic Unit

P Phosphorous/ Phosphate

PCR Polymerase Chain Reaction

PGPR Plant Growth Promoting Rhizobacteria

pH Hydrogen ion concentration

PRSS Pulses Research Sub-Station

ppm Parts per million

PSB Phosphate Solubilizing Bacteria

RCBD Randomized Complete Block Design

RMM Rhizobial Minimal Medium

RNA Ribonucleic Acid

rpm Revolution per minute

rRNA Ribosomal RNA

SDS Sodium Dodecyl Sulfate

Taq Thermus aquaticus

TE buffer Tris EDTA buffer

Temp Temperature

TCP Tri-Calcium Phosphate

U Units

UV Ultra Violet

YMA Yeast Mannitol Agar

TY Tryptone Yeast

http://www.aari.punjab.gov.pk/institutes-sections/pulses-research-institute/puri-allied-stations/pulses-research-sub-station-kallurkot

1

1. Introduction

1.1 Chickpea Crop

Chickpea (Cicer arietinum L.) is a very good source of proteins, carbohydrates,

minerals (calcium and iron) and vitamins [1]. After beans, it is the second most

important cultivated legume which fulfills the protein requirement of the people living

in developing countries [2]. India typically produces 2/3rd of the world chickpea output.

Pakistan stands second in the context but it’s yield is very low i.e., 500 kg per hectare.

Most of the Pakistani farmers are resource-poor and cannot afford to apply

recommended doses of fertilizers due to high prices. There are two main types of

chickpea; the “Desi-type” and the “Kabuli-type”. The former is small seeded, with a

colored testa and angular shape while the latter is large-seeded and beige colored

(Figure 1.1). More than 80 % of the world production is of Desi-type, predominantly

grown in subsistence agriculture regions. According to Economic survey of Pakistan

(2012-2013), chickpea is the largest Rabi pulse in the country, with the annual

production of 0.574 million tons.

Figure 0-1 Seeds and flowers of Desi, Kabuli-type chickpea

1. Introduction

2

1.2 The Chickpea-Mesorhizobium Symbiosis

Despite the fact that chickpea is very important legume in term of nutritional value,

area under cultivation and yield, chickpea-rhizobial symbiosis has not been extensively

studied due to its predominant cultivation in underdeveloped countries [3]. Legume-

rhizobial symbiosis depends on the specificity of legume and rhizobial species which

results in the formation of specialized root structures called “nodules”. The nodules

host rhizobial cells which reduce atmospheric nitrogen to ammonium [4]. This

biological nitrogen (N) fixation fulfills 80-90 % of N requirements of legumes.

Biological nitrogen fixation in chickpea may range 0-176 kg / ha of N, depending upon

rhizobial strain and the environmental conditions [5]. Chickpea responds positively to

inoculation and results in improved N fixation and yield [5]. Only the member of genus

Mesorhizobium can effectively nodulate chickpea and the genus has been revised to

accommodate some species previously included in the Rhizobium genus [3, 6].

Presently the genus Mesorhizobium includes 41 species of which 8 mesorhizobial

species namely M. amorphae, M. ciceri, M. huakuii, M. loti, M. mediterraneum, M.

muleiense, M. opportunistum, and M. tianshanense have been identified as bacteria

capable of nodulating chickpea [3]. However, only three mesorhizobial species namely

M. ciceri, M. mediterraneum and M. muleiense have chickpea origin [3].

Mesorhizobium sps. have been reported to induce nodulation, enhance nutrient up-take

as well as increase chlorophyll contents of chickpea [7]. Moreover, it has been reported

that chickpea nodulated with Mesorhizobium spp. shapes the rhizospheric soil

microbiome and helps to improve the establishment of subsequent crops [8].

1.3 Bacterial Diversity Associated with the Root Nodules

Legumes, including chickpea, produce root nodules to accommodate nitrogen-fixing

symbiotic bacteria collectively called “rhizobia”. Traditionally, rhizobia were classified

as α-Proteobacteria belonging to the genera Allorhizobium, Azorhizobium,

Bradyrhizobium, Ensifer (Sinorhizobium), Mesorhizobium and Rhizobium [9]. In

addition to well-known rhizobia, several root nodule inducing bacteria have also been

described in legumes including α-Proteobacteria (Devosia, Methylobacterium,

Microvirga, Ochrobactrum and Phyllobacterium) and β-Proteobacteria (Burkholderia,

Cupriavidus and Ralstonia) [9]. In addition to rhizobial strains that can induce nodules

effectively, other bacterial species incapable of host nodulation have also been reported

1. Introduction

3

from legume nodules. These bacteria are commonly known as Non-Rhizobial

Endophytes (NRE) [10]. NRE include member of three classes α-, β- and γ-

Proteobacteria as well as some Actinobacteria and Firmibacteria [9-14]. NRE genera

of α-Proteobacteria include Aminobacter, Ancylobacter, Bosea, Caulobacter, Devosia,

Inquilinus, Methylobacterium, Novosphingobium, Ochrobactrum, Paracoccus,

Phyllobacterium, Shinella, Sphingomonas and Tardiphaga. Among the NRE, β-

Proteobacteria are represented by the genera Bordetella, Duganella, Herbaspirillum,

Massilia and Variovorax. In the class γ-Proteobacteria, genera Acinetobacter,

Buttiauxella, Enterobacter, Pantoea, Pseudomonas, Serratia and Stenotrophomonas

are considered important members of NRE. Actinobacteria like Arthrobacter,

Curtobacterium, Kocuria, Kribbella, Microbacterium, Mycobacterium, Nocardia and

Streptomyces are the representative genera in NRE. In addition, Firmibacteria of the

genera Bacillus, Brevibacillus, Cohnella, Lysinibacillus, Paenibacillus and

Staphylococcus are also included in NRE. Multiple modes of action have been proposed

for plant growth promotion by NRE as well as free-living plant growth promoting

rhizobacteria (PGPR). This shows that root nodule endophytes include (i) true

endosymbionts that fix atmospheric nitrogen for plant use (ii) “helper bacteria” promote

nodulation process when co-inoculated with true endosymbionts and (iii) opportunistic

endophytes that avail nutrient (nitrogen) rich nodule environment [9, 15].

1.4 The Rhizospheric Soil

Rhizospheric soil is defined as a specialized soil environment under the influence of

living-roots where complex microbial communities are supported by root exudates,

mucilage and sloughed-off root cells. The chemistry and rhizodeposition of root

exudates defines the microbial ecology on the roots and in the surrounding soil. Soil

microbes in turn influence the composition and quantity of various root exudates

components through their effects on root cell leakage, cell metabolism, and plant

nutrition [16, 17]. Rhizospheric soil is also the site of intense microbial activity and

responsible for nutrient mobilization for the plant [18]. Soil microorganisms are

involved in cycling of basic elements such as carbon, phosphorous, nitrogen, sulphur

and micronutrients and have role in plant nutrition, plant health, soil structure and soil

fertility [19].

1. Introduction

4

1.4.1 Identification of Bacterial Isolates from Rhizospheric Soil

Identification and characterization of the bacteria is an important component of

bacterial diversity. Bacteria can be identified on the basis of phenotypic and

biochemical chracteristic and genetic methods like DNA-DNA hybridization and 16S

rRNA gene sequence analysis [20]. Traditionally bacterial isolates from rhizospheric

soil have been identified by studying morphological, biochemical and physiological

characters of bacterial isolates. However, very small proportion of bacterial species are

culturable. Even after more than 100 years of pure culture studies, knowledge of

bacterial diversity obtained is still considered incomplete [20, 21]. Furthermore,

phenotypic properties of cultureable bacterial isolates are not always reproducible and

the isolation procedure varies from laboratory to laboratory e.g., growth media used,

purification and storage procedures [20, 22].

Among the DNA based techniques, 16S rRNA sequence analysis has been

frequently used for the identification and to study phylogenetic relationship of bacteria.

Among the most important characteristics of 16S rRNA gene are its universal

distribution, highly conserved structures, and relative evolutionary stability essential

for analyzing high level (kingdom, family etc) taxonomic relationships and evolution

of the gene at a relatively constant rate over time. Another gene commonly used for

studying diversity of nitrogen fixing bacteria is nifH which is highly conserved among

the structural genes of nitrogenase enzyme [20, 23, 24].

1.5 Mechanisms used by Bacteria for Plant Growth

Promotion

Free-living bacteria present in the rhizospheric soil of plants which stimulate plant

growth collectively are known as Plant Growth Promoting Rhizobacteria (PGPR).

PGPR exhibit plant-beneficial traits and utilize one or more than one mechanisms for

plant growth promotion, including nitrogen-fixation, P-solubilization and

phytohormone production. PGPR include diverse bacterial genera like Acetobacter,

Acinetobacter, Arthrobacter, Azoarcus, Azospirillum, Azotobacter, Bacillus,

Burkholderia, Enterobacter, Herbaspirillum, Klebsiella, Ochrobactrum, Pantoae,

Pseudomonas, Rhodococcus, Serratia and Zoogloea [25]. Following are the direct

mechanisms utilized by PGPR: i) atmospheric nitrogen fixation ii) phytohormones

production such as auxins, cytokinins, gibberellins iii) sequestering of iron (Fe) by

1. Introduction

5

production of siderophores iv) phosphate solubilization and v) lowering of ethylene

concentration due to ACC deaminase activity [25-27]. The indirect mechanisms of

PGPR may prevent the harmful effects of plant pathogens by production of inhibitory

substances or by increasing the natural resistance of the host [26]. Enormous efforts are

being made to understand the ecology of PGPR, yet their development as inoculants

remains a major challenge.

1.5.1 Biological Nitrogen Fixation and Nodulation

Symbiosis between rhizobial bacteria and leguminous crop leads to the formation of

the root nodules. Biological nitrogen fixation takes place in the root nodules. Several

nitrogen fixation genes have been identified for the role in the synthesis of nitrogenase

and catalysis. Among the structural (nif) genes, nifH is standard gene for identification

of diazotrophs as it is highly conserved among the nitrogen fixation genes. Due to

conserved nature of nifH gene, it has been frequently used as a detection tool for

screening the presence of diazotrophs [28]. For the formation of root nodule, host

compatible rhizobia attach to the tip of growing root hairs of legume plant, initiate tube-

like structure called the infection thread, which grows toward the root cortex and

transports the multiplying bacteria. During the infection, cell cycle activation in the

cortical cell leads to cell proliferation and formation of the nodule primordium. When

the infection threads reach the primordium, the rhizobia are released as membrane-

bound droplets in the legume plant cells, where development of symbiotic cells and

nodule differentiation begins [29]. Concentration of oxygen in the nodule is particularly

important given the fact that nitrogenase activity is highly inhibited by oxygen [30].

Leguminous plants produce oxygen carrier compound “leghemoglobin”.

Leghemoglobin maintains the concentration of oxygen in which biological nitrogen

fixation performs well [31]. Legume host provides the carbon and energy sources

(sucrose and dicarboxylic acids) to nodules to fix atmospheric N [32]. Chickpea like

other legumes fulfills the N requirement by symbiosis with Mesorhizobium spp. and

this association is very critical for the growth and yield of chickpea [33].

In addition to symbiotic rhizobia, free-living N-fixing bacteria have been also

isolated from the rhizosperic soil of different plants. Free-living N-fixing bacteria

belong to diverse genera e.g., Arthrobacter, Azoarcus, Azospirillum, Azotobacter,

Bacillus Enterobacter, Herbaspirillum, Klebsiella, Pseudomonas and Zoogloea etc [34,

1. Introduction

6

35]. Due to effort being made to reduce the impact of chemical nitrogen fertilizers,

interest in the nitrogen fixing PGPR has increased recently [36].

1.5.2 Phytohormone Production

Phytohormones such as auxins, cytokinins and gibberellins produced by PGPR may

also aid in growth and development of host plant species. Among the phytohormones,

production of auxins by a wide, diverse group of soil microbial isolates has been

demonstrated [37]. The production of IAA by PGPR has been taken as the main

criterion in the selection of PGPRs for the seed and seedling treatments [38].

Phytohormone IAA contributes to the plant growth by playing a role in: i) cell

enlargement, ii) cell division, iii) root initiation, iv) root growth inhibition, v) increased

growth rate, vi) phototropism, vii) geotropism and viii) apical dominance [39]. About

80 % microorganisms isolated from the rhizospheric soil of various crops have the

ability to produce auxins (IAA) as secondary metabolites [39, 40]. A number of

bacterial isolates like Aeromonas, Azospirillum, Azotobacter, Enterobacter, Klebsiella

and Pseudomonas have been reported to produce the auxin in the liquid medium

containing tryptophan [41]. The IAA producer strains have been purified from the

rhizospheric soil of important crops like sugarcane, wheat and rice and production of

IAA has been demonstrated in growth media containing tryptophan [42-44]. A number

of bacterial strains i.e., Mesorhizobium, Ochrobactrum, Pseudomonas, Rhizobium and

Serratia isolated from the root nodules of chickpea produced the IAA in the liquid

media and also have been shown to have positive effect on the growth of chickpea [33,

45-47]. Similarly IAA producing isolates from rhizosphere soil like Azotobacter,

Bacillus, Chryseobacterium, Enterobacter, Pantoea, Pseudomonas, Rhizobium,

Serratia, Sphingobacterium and Streptomyces also have positive effect on the yield of

chickpea [48-54].

The presence of auxins in soil may have an environmental impact affecting plant

growth and development. IAA is a major microbial metabolite derived from L-TRP and

detected in soil by use of high performance liquid chromatography (HPLC). Phenotypic

character of the soil microbiota has more of an influence on auxin production than the

soil physicochemical properties (e.g., pH, organic C content, CEC, etc.).[55, 56].

1. Introduction

7

1.5.3 Phosphate Solubilization

Phosphorus is another essential macronutrient for crops after nitrogen [57]. Most of the

phosphorous in soil is present in form of insoluble chemical complexes of phosphate

ions with iron, magnesium, aluminum and calcium. Plants are not able to utilize

insoluble phosphorous from soil. Therefore, soils are often supplemented with

inorganic phosphorus as chemical fertilizers to support crop production. However,

repeated use of chemical fertilizers deteriorates soil quality [58]. Therefore, the present

scenario is shifting towards the use of PGPR as biofertilizers. Soil microbes are an

integral part of the soil phosphorus cycle and convert insoluble phosphorous into

soluble form to be taken up by plants. Microorganisms and their interactions in soil

mediate the distribution of phosphate between the available pool in soil solution and

the total soil phosphorus through solubilization and mineralization reactions, and

through immobilization of phosphate into microbial biomass and/or formation of

sparingly available forms of inorganic and organic soil phosphate [35, 59]. Soil bacteria

with P-solubilizing capacity are present in the plant rhizospheric soil and are called

Phosphate Solubilizing Microorganisms (PSM) or Phosphate Solubilizing Bacteria

(PSB) [60]. PSB play a vital role in increasing the phosphorus uptake, crop yield, as

well as induce resistance against salinity and pathogens [25, 61, 62].

Application of PSB increases soil fertility due to their ability to convert

insoluble phosphorus to soluble phosphorus by releasing organic acids, chelation and

ion exchange [63]. The use of P-solubilizing microbes in agriculture can favour a

reduction in agro-chemical use and support eco-friendly crop production [60, 64]. P-

solubilizer bacterial isolates from chickpea root nodule and rhizosphere soil like

Azotobacter, Bacillus, Chryseobacterium, Enterobacter, Mesorhizobium

Pantoea, Pseudomonas, Rhizobium, Serratia, Sphingobacterium and Streptomyces

have been reported to promote the growth of host when applied as single inocula or co-

inoculated with Mesorhizobium [33, 48-54, 65]. Production of different organic acids

e.g., gluconic, oxalic, 2-ketogluconic, lactic, succinic, formic, citric and malic by PSB

in the pure cultures for the solubilization of tricalcium phosphate has been reported.

Based on profiling of organic acids produced in pure culture, cluster analysis revealed

inter-species and intra-species variations [66, 67].

http://link.springer.com/article/10.1007/s11738-016-2284-6

1. Introduction

8

1.6 Application of Bacterial Inoculation for Plant Growth

Promotion Legume inoculation is an old practice that has been adopted for more than a century in

agricultural systems [68]. Bacterial inoculants which promote plant growth are

generally considered to be of two types a) symbiotic and b) free-living [27, 69].

Chickpea responds positively to inoculation with compatible rhizobia (Mesorhizobium

spp.) and results in improved N fixation, chlorophyll contents and yield [3, 5, 7].

Moreover, it has been reported that chickpea shapes the rhizospheric soil microbiome

and helps to improve the establishment of subsequent crops [8]. Single inocula of

mesorhizobia or co-inoculation of Mesorhizobium with Azotobacter, Bacillus and

Pseudomonas has resulted in improved nodulation, growth and yield of chickpea [44,

70, 71].

In addition to symbiotic rhizobia, free-living PGPR can also be used as bio-

inoculants for crops. It has been reported that that inoculation with free-living P-

solubilizer bacteria e.g., Bacillus, Pseudomonas and Serratia has increased the crop

yields [60]. Inoculation of soil with P-solubilizing bacteria is a promising approach that

may alleviate the deficiency of phosphorus. This bioavailability of soil inorganic

phosphorus in the rhizospheric soil varies considerably with plant species and

nutritional status of soil. It has been reported that co-inoculation with P-solubilizing

bacteria and nitrogen fixing Rhizobium stimulated plant growth more profoundly than

their separate inoculations [72]. Improvement in the chickpea nodulation,

photosynthetic rate, transpiration rate, nutrient up-take, microbial biomass C and

growth has been reported recently by integrated use of Mesorhizobium ciceri, PGPR

along with P-enriched compost under irrigated as well as rainfed farming systems of

Pakistan [73]. Application of biofertilizers based on rhizobia alone as well as co-

inoculation of PGPR along with endophytic rhizobia have resulted in improved growth

of chickpea [60].

1.7 Metagenomics

Bacterial populations in complex communities such as soil can be studied by (i)

isolation and identification of the microorganisms inhabiting the community and (ii)

determination of the various functions carried out by the microbes. Traditionally,

microbiologists first isolated pure cultures (or co-cultures) of microorganisms from the

1. Introduction

9

environment followed by an analysis of their physiological and biochemical traits. The

major drawback of this approach is the inability to obtain the majority of the microbes

in pure culture from complex environment samples like soil. To quantify active cells in

environmental samples, viable cell counts and most-probable-number techniques are

frequently used [22]. However, these methods select for only a small percentage of

dominant organisms and frequently fail to provide information about majority of the

microbes that make up these communities.

For efficient use of microbes for plant growth promotion, information about

composition of bacterial communities in the root zone is a pre-requisite. However, only

limited information about composition of soil microbial communities can be made

available through traditional culture-dependent techniques as only 0.1 to 10 % of soil

bacteria are accessible through these technologies. Therefore, culture-independent

techniques i.e., extraction of DNA directly from soil followed by PCR amplification

and sequence analysis of genes are being frequently employed for the investigation of

bacterial diversity in soil and environmental samples [74]. The information on

microbial diversity and community structure of soil assembled through high-throughput

pyrosequencing of soil DNA is also important for agriculture as microorganisms are

playing fundamental roles in biogeochemical cycles that determine plant health and

nutrition [75, 76]. In 1998, Jo Handelsman coined the term “metagenomics” to describe

analyses of genetic materials recovered directly from environmental samples [75].

Metagenomics includes research involving the application of modern genomic DNA-

based techniques to the study of biological communities directly in their environments,

by avoiding the need for isolation, lab cultivation and observation of individual

organisms [23]. 16S rRNA gene has been the marker of choice for metagenomics

studies [77]. Another gene commonly used for studying diversity of nitrogen fixing

bacteria is nifH which is highly conserved among the structural genes of nitrogenase

enzyme [24, 78]. 16S rRNA gene sequences analysis of Arabidopsis thaliana associated

bacteria communities has revealed that the host genotype and soil type strongly

influenced the bacterial communities [79]. Another study on effect of conventional and

organic systems on bulk soil bacterial communities evaluated by pyrosequencing

revealed that Proteobacteria were abundant in organic farming and

Actinobacteria were abundant in conventional farming [80]. 16S rRNA gene

pyrosequencing of wheat rhizosheath DNA showed that 57 % of all the genera detected

1. Introduction

10

through retrieved sequences were commonly found in both wheat-cotton and wheat-

rice rotations while 19.4 % were present only in wheat rice rotation and 8.3 % were

uniquely present in wheat-cotton rotation [81]. The NRE of legumes have been poorly

studied compared to symbiotic bacteria [82]. There is no report regarding bacterial

diversity of nodule of chickpea by culture-independent DNA based technique.

Therefore, pyrosequencing of 16S rRNA and nifH genes directly amplified from root

nodule and rhizospheric DNA as well as cultivation of root nodule and rhizospheric-

associated bacteria was attempted in the present study to investigate bacterial diversity

in root nodules and rhizospheric soil of chickpea.

1.8 Objectives of the Present Study

1. To study bacterial diversity in the nodules and rhizospheric soil of chickpea

varieties growing in different regions of Pakistan

2. To study the effect of bacterial inoculations (nitrogen-fixers, phosphate

solubilizers and phytohormone producers) on plant growth

3. To study nodule occupancy and survival of inoculated bacteria in the

rhizospheric soil

11

2 Materials and Methods

The experimental work reported in thesis was conducted at Plant Microbiology Group

NIBGE Faisalabad Pakistan, Microbial Genetics Group University of Geneva

Switzerland and Utah Water Research Laboratory Utah State University USA .

2.1 Sample Collection

Rhizospheric soil and root nodules of chickpea were collected from different chickpea

growing areas of Pakistan (Figure 2.1) including NIFA, District Peshawar

(34°00'55.9"N 71°42'46.3"E), Kallar Syedan, District Rawalpindi (33°22'37.8"N

73°30'22.4"E), Silanwalli, District Sargodha (31°42'17.9"N 72°22'56.3"E), Thal desert,

District Bhakkar, Jhang and Khushab (30°44'29.6"N to 32°14'12.8"N 71°10'07.3"E to

72°06'48.3"E), Chowk Munda, District Muzaffargarh (30°35'04.3"N 71°14'55.2"E),

Chichawatni, District Sahiwal (30°30'10.4"N 72°45'33.8"E), NIAB and NIBGE,

District Faisalabad (31°23'42.5"N 73°01'45.5"E). Samples were collected by marking

circle radius of about ≈15 cm around the plant and dug up to depth of ≈20 cm using a

spade. The plants were uprooted by carefully removing the soil clump with intact root

system. Soil was removed carefully by avoiding the detachment of secondary roots.

Nodules were detached by cutting roots 0.5 cm away from each side of nodule and

desiccated in glass vials containing silica gel. The rhizospheric soil samples (i.e., soil

attached with roots) were collected in plastic bags and thoroughly homogenized before

further processing in the lab.

2.2 Soil Analysis The soil samples were air dried, sieved through 2 mm sieve and analysed for soil texture,

pH, cation exchange capacity (CEC), total nitrogen (N) total phosphate (P), available P

and potassium (K). Detailed standard analysis procedure were the same as reported

previously [83-89].

https://www.researchgate.net/institution/Utah_State_University/department/Utah_Water_Research_Laboratory

https://www.researchgate.net/institution/Utah_State_University

2. Materials and Methods

12

Figure 2-1 Map of Pakistan showing different sampling locations

2.3 Bacterial Isolation

2.3.1 Isolation of Endophytic Bacteria from Nodules of Chickpea

Nodules were detached from the roots and washed gently under running tap water to

remove soil particles. Undamaged nodules were washed in 70 % ethanol for 10 min

then rinsed three times in sterilized water. Nodules were surface sterilized with 5 %

hydrogen peroxide or 4 % sodium hypochlorite for 10 min and rinsed 5 times with

sterilized water. Nodules were cut with a sterilized knife or crushed and then a loop full

of nodule sap was streaked on Yeast Manitol Agar (YMA) medium [90], Tryptone

Yeast (TY) medium [91] and Rhizobial Minimal Medium (RMM) [92] medium

Petriplates. Plates were incubated at 28 °C until the appearance of bacterial colonies.

The bacteria were purified by repeated sub-culturing of single colonies. The colonies

were re-purified by serial dilution using RMM medium.

For isolation of specifically Serratia strains from chickpea root nodules, surface

sterilized nodules were crushed and a loop full of nodule sap was streaked on Luria-

Bertani (LB) medium [93] agar Petriplates. Plates were incubated at 30 °C for 2-4 days.


13

Red coloured colonies, typical of Serratia strains, were purified by repeated sub-

culturing of single colonies.

Gram’s staining was done according to Vincent method [94]. The cell

morphology and motility of bacterial strains were observed under light microscope

(Labophot 2, Nikon, Japan).

2.3.2 Isolation of PGPR from Rhizospheric Soil of Chickpea

Three culturing media i.e., Luria-Bertani (LB) medium [93], Nitrogen Free Malate

(NFM) medium [95] and Pikovskaya medium [96] were used for the isolation and

purification of bacteria present in the rhizospheric soil of chickpea. Rhizospheric soil

attached with the roots was carefully removed from the plants, mixed thoroughly and

then one gram representative soil sample was taken. The soil samples were used for

preparation of serial dilution (10X) according to the method described by Somasegaran

and Hoben [97]. 100 µL of dilutions from 10-4 to 10-6 were spread on LB, YMA and

Pikoviskya media agar plates. Aliquots (100 µL) from 10-3 to 10-6 were inoculated in

semi-solid NFM medium vials.

The plates were incubated for different time periods (1-7 days) at 30 ºC and

morphologically different colonies appearing on the growth media were selected for

further purifications. The bacteria were purified by repeated sub-culturing of single

colonies.

The bacterial colonies were counted and number of cells per gram of soil was

calculated according to the method described by Somasegaran and Hoben [97]. The

NFM medium vials were kept for further purification of nitrogen fixing bacterial

fraction of the rhizospheric soil. For purification and enrichment of cultures, 50 µL

from each tube were transferred to fresh Eppendorf tubes containing NFM medium

after 48 hours and the procedure was repeated up to 5-6 times. Enrichment cultures

were streaked on NFM medium agar plates to get single colonies. Single colonies

were again inoculated in semi-solid NFM medium as well as to LB broth and LB

agar plates at 30 ºC for 1 to 3 days and used for studying colony and cell-morphology.

Gram’s staining was done according to Vincent method [94]. The cell morphology and

motility of bacterial strains were observed under light microscope (Labophot 2, Nikon,

Japan).


14

2.4 Molecular Characterization and Identification of

Bacterial Isolates

2.4.1 Extraction of Genomic DNA from Pure Cultures

Total genomic DNA of bacterial strains was extracted by cetyl trimethyl ammonium

bromide (CTAB) method [98] with slight modifications. Overnight grown bacterial

cultures (1.5 mL) in LB medium at 30 °C were used for DNA extraction. Cells

harvested by centrifugation at 12000 x g, were re-suspended in 567 µL of TE (Tris 10

mM; EDTA 1 mM) buffer, lysed with the 3 µL of proteinase K (10 mg/mL) and 30 µL

of 10 % SDS, followed by incubation at 30 °C for an hour to allow complete lysis. 100

µL of 5 M NaCl and 80 µL of CTAB/NaCl (10 % CTAB; 0.7 M NaCl) solutions were

added and the lysate was mixed thoroughly and incubated at 65 °C for 10 min. DNA

was purified by sequential phenol, phenol-chloroform, and chloroform extractions,

followed by isopropanol precipitation. The pellets were washed with 70 % ethanol and

re-suspended in 100 µL of TE buffer. The samples were stored at -20 ºC until use.

2.4.2 Identification by nifH and 16S rRNA Gene Amplification

DNA extracted from pure cultures was used for nifH and 16S rRNA gene amplification

using nifH PCR primers POL F (5′-TGCGAYCCSAARGCBGACTC-3′) and POL R

(5′-ATSGCCATCATYTCRCCGGA-3′) [24] and 16S rRNA PCR primers PA (5′-

AGAGTTTGATCCTGGCTCAG-3′) and PH (5′-AAGGAGGTGATCCAGCCGCA-

3′) [99]. A 25 µL PCR reaction volume containing 0.2 mM of each dNTPs, 0.5 μM of

each primer, 2.5 µL of 10X PCR buffer, 25 mM of MgCl2, 20 ng of template DNA, and

0.15 U of Taq DNA polymerase (Fermentas, Germany) was used. The PCR conditions

were 5 min of denaturation at 94 °C, followed by 35 rounds of temperature cycling; 95

°C for 30 s, 52 °C for 30 s, and 72 °C for 45 s and a single step final extension at 72 °C

for 7 min. PCR products were eluted using gel extraction kit (Fermentas, Germany) and

sent for commercial sequencing (Macrogen Inc., South Korea).

2.4.3 Phylogenetic Analysis of the Bacterial Isolates

16S rRNA and nifH gene from pure cultures and their representative type strain

sequences from NCBI database were aligned using Clustal X [100] and maximum

likelihood (ML) based phylogenetic tree was constructed using MEGA (version 6)

[101]. Confidence in the tree topology was evaluated using bootstrap re-sampling

methods (1000 replications) and bootstrap values of at least 50 %, which demonstrate


15

good support measures were retained. Ensifer sojae CCBAU 05684T (GU994077)

(only for nifH gene), Escherichia coli ATCC 11775T (X80725), Azorhizobium

oxalatiphilum DSM 18749T (FR799325) and Pseudomonas aeruginosa DSM 50071T

sequence were used as an outgroup.

2.5 Preservation of Bacteria

The bacterial isolates were grown at 27 °C to 30 °C to get an optical density at 600

nm (OD600) 0.4 to 0.7 and preserved in glycerol (20 %) at -80 °C.

2.6 Characterization of Bacterial Isolates

2.6.1 Confirmation of Nodulation Ability by Endophytic Bacterial

Isolates on Chickpea

Seeds of two chickpea varieties, i.e., Punjab 2008 (Desi-type) and Noor 2009 (Kabuli-

type), were obtained from Pulses Research Institute, Ayub Agricultural Research

Institute, Faisalabad. Seeds were surface-sterilized with 70 % ethanol for 10 min, and

then with 5 % hydrogen peroxide for 1 min, followed by subsequent washings with

sterilized distilled water and sown in plastic jars containing one kg sterilized sand. For

inoculum preparation the bacterial isolates were grown in 100 mL of TY medium and

incubated at 28+2 ºC to get an optical density at 600 nm (OD600) 0.4 to 0.7 (about

1X109 cfu/mL). Cultures were centrifuged at 10,000 x g for 10 min. The cell pellet was

washed with 0.85 % saline solution and re-suspended in 100 mL of saline. The

seedlings were inoculated with 1 mL bacterial suspension (1X106 cfu/mL) at the time

of germination. Pots were irrigated according to the requirement of plants with

sterilized water. Plants were harvested after 42 days of inoculation. Data on number of

nodules, dry weight of nodules and shoot were recorded.

2.6.2 Indole-3-Acetic Acid (IAA) Production by Isolates

For detection and quantification of indole-3-acetic acid (IAA) production by bacterial

isolated, cultures were grown at 20 ºC, 30 ºC and 40 ºC for 7 days in LB medium

supplemented with L-tryptophan (100 mg/L). The supernatant was obtained from cell-

free culture by centrifugation at 12000 x g for 10 min and the pH was adjusted to 2.8

with hydrochloric acid (1N). Initially screening was done by Qualitative Test/Spot

Test as described by Gordon and Weber [102] with the following modifications. 100


16

µL of each bacterial culture was mixed with 100 µL of Salkowski reagents (1 mL 0.5

M ferric chloride, 30 mL sulphuric acid with specific gravity 1.84 and distilled water

50 mL) in ELISA plate. 100 µL IAA (20, 10, 5 and 0 ppm) was used as standard and

mixed with equal volume of Salkowski reagent. The tubes were visualized after 20

min to 75 min for purple, pink or purplish-pink colour development. Development of

pink colour indicated the IAA producing ability of bacterial strain.

Bacterial strains were further investigated for quantitative analysis of IAA.

IAA was extracted from the acidified spent medium with equal volumes of ethyl

acetate [103], evaporated to dryness and re-suspended in 1.0 ml of methanol. The

samples were analyzed by HPLC (Varian Pro star) using a UV detector and a C-18

column as described previously [104].

2.6.3 Phosphate Solubilization by the Bacterial Isolates in Pure

Culture

Qualitative Method

Initially screening of isolates was done by qualitative method. Bacterial cultures were

grown in LB or TY (LB was used for free-living and TY was used for endophytes)

broth media to get an optical density at 600 nm (OD600) 0.4 to 0.7 and 10 µL of this

culture was dropped on Pikovskaya agar plate [96]. The plates were incubated at 30 °C

for 15 days. The appearance of a zone of halo on the plates indicated phosphate

solubilization activity of bacterial strains.

Quantitative Method

Bacterial cultures were grown in 100 mL of Pikovskaya broth medium for P-

solubilization for 15 days at three incubation temperatures i.e., 20 ºC, 30 ºC and 40 ºC

and constant shaking at 120 revolutions per min (Kuhner shaker, Switzerland). Flasks,

containing the same medium but without inoculation, were treated as blank. Cell-free

supernatant was obtained by centrifuging at 12000 X g for 10 min. This cell-free

supernatant was filtered with 0.2 µm filter (Orange Scientific GyroDisc CA-PC,

Belgium) to remove residues. Cell-free supernatant was used for measuring soluble P

by Mo-blue (molybdate blue) colour method on spectrophotometer (Camspec M350

double beam UV visible, UK) at 882 nm [105, 106]. For determination of solubilized

P, standard curve of KH2PO4 using 2, 4, 6, 8, 10, 12 ppm solutions was prepared.


17

2.6.4 Organic Acid Production by the Bacterial Isolates in Pure

Culture

For detection and quantification of organic acids produced for P solubilization selected

bacterial strains were grown in 100 mL Pikovskaya broth medium for 15 days at 30 ºC

temperature and constant shaking at 120 revolutions per min (Kuhner shaker,

Switzerland). Cell-free supernatant was obtained by centrifuging at 12000 X g for 10

min and filtered with 0.2 µm filter (Orange Scientific GyroDisc CA-PC, Belgium) to

remove residues. Cell-free supernatants (10 mL) of bacterial strains were concentrated

to 1.5 mL in a concentrator (Eppendorf Concentrator 5301, Germany) and again filtered

using 0.2 µm filter. The samples were analyzed on HPLC as described previously [107].

2.7 Effect of Bacterial Inoculations on Chickpea

2.7.1 Earthen Pot Experiments to Study the Effect of Bacterial

Inoculation on Chickpea

Two chickpea varieties, i.e., Punjab 2008 (Desi-type) and Noor 2009 (Kabuli-type),

were grown in earthen pots containing 20 kg of non-sterilized soil (sandy loam, EC

2.5 ds/m, pH 8.2, organic matter 0.6 %, available P 7.5 mg/kg and total N 0.06 %).

Seeds were sterilized with 70 % ethanol for 10 min then rinsed 5 times with sterilized

water. Three plants were maintained in each pot with five replicates. For preparation

of inoculum the bacterial isolates were grown at 30 °C to reach an optical density

at 600 nm (OD600) 0.4 to 0.7 (about 1X109 cfu/mL) in 100 mL of LB medium or TY

medium (for endophytes) and centrifuged at 10,000 x g for 10 min. The cell pellet

was washed with 0.85 % saline solution and re-suspended in 100 mL of saline.

Bacterial consortium was prepared by mixing of equal volume of bacterial cultures.

200 µL were added to each seedling in the inoculated treatments. 200 µL of saline

were used for non-inoculated control. Pots were irrigated according to the requirement

of plants. Number of nodules and dry weight of nodules were recorded at flowering

stage. Plants were harvested after maturation and data on grain weight and straw

weight were recorded.

2.7.2 Field Trials to Study the Effect of Bacterial Inoculation on

Chickpea

Two chickpea varieties, i.e., Punjab 2008 (Desi-type) and Noor 2009 (Kabuli-type),

were used in field experiments conducted at NIBGE and AARI experimental field


18

(District Faisalabad), AZRI, Bhakkar and PRSS, Kalurkot located in the Thal desert

(District Bhakkar). Location and other characteristics of all sites are given in the

results section Table 3.19. At the experimental field of NIBGE and AARI, soil was

properly prepared by ploughing at optimum moisture contents after applying canal

water for irrigation. The sowing at the Thal desert was done on natural (rain water)

preserved moisture. We used a randomized complete block design and plot size was 4

m X 4 m at all sites. For preparation of inocula, bacterial isolates were grown at 30 °C

to an optical density at 600 nm (OD600) 0.4 to 0.7 (about 1X109 cfu/mL) in 100 mL of

LB or TY medium (for rhizobia) and centrifuged at 10,000 x g for 10 min. The cell

pellet was washed and re-suspended in 100 mL 0.85 % saline solution. Seeds were

added to cell suspension along with autoclaved powdered filter-mud as carrier

material and sown in the randomized block design. For non-inoculated (control)

treatment only saline solution along with autoclaved filter-mud was used for seed

coating. Number and dry weight of nodules were recorded at flowering stage. Plants

were harvested after maturation. Data on grain weight and straw weight were recorded.

In case of Serratia inoculation, before sowing half blocks were fertilized with 10 g

diammonium phosphate (DAP) (equivalent to half of the recommended dose) and no

fertilizer was applied to the remaining plots. Recommended fertilizer dose for

chickpea is 25-50-25 kg NPK/ha [108].

2.8 Bacterial Diversity in Rhizospheric Soil and Root

Nodules Studied by Culture-Independent DNA-Based

(16S rRNA and nifH Genes Sequences) Method For extraction of DNA, nodules were washed with 70 % ethanol for 10 min and washed

3 times with sterilized water. The nodules were surface-sterilized with 5 % hydrogen

peroxide for 5 min and washed 5 times with sterilized water. DNA extractions were

made by mechanical lysis using “Bead Beater” and DNA isolation kit (MP biomedical,

USA) according to the manufacturer’s instructions.

The extracted DNA samples were amplified with sequencing primers F515 (5′-

GTGCCAGCMGCCGCGG-3′), R907 (5′-CCGTCAATTCMTTTRAGTTT-3′) for

16S rRNA gene and POL F (5′-TGCGAYCCSAARGCBGACTC-3′) and POL R (5′-

ATSGCCATCATYTCRCCGGA-3′) for nifH gene, which were attached with unique

identifier and adopter sequences. The detailed PCR conditions for amplicon sequencing

were the same as described previously [109]. Amplified PCR products were purified


19

with Agencourt AMPure beads (Beckman Coulter, Brea, CA). Purified PCR products

from different samples were pooled in equimolar concentrations. Pyrosequencing was

performed on the mixture with the 454 GS FLX sequencer (454 LifeSciences) at the

Utah State University Center for Integrated Biosystems.

Overall bacterial 16S rRNA gene sequences amplified from the root nodules of

two chickpea types from 5 different localities were processed as described previously

[109]. All good quality sequences were identified through Ribosomal Database Project

(RDP; http://rdp.cme.msu.edu), Naive Bayesian Classifier 2.5 [110]. For the

Assessment of the Serratia species associated with chickpea root nodules by 16S rRNA

gene barcoded pyrosequencing were retrieved using Mothur [111]. Serratia related

sequences (1136) from root nodules and two pure culture isolates sequences of different

Serratia species were aligned using MUSCLE [112] and clustered in operational

taxonomic units (OTUs) at 99 % DNA identity.

2.9 Statistical Analysis

Effect of bacterial inoculations on different growth parameter of chickpea was

determined through 4-way analysis of variance (ANOVA) using Statistix 8.1 software.

Mean values were compared by applying least significant difference test (LSD) at alpha

0.05 on all the parameters.

Overall geochemical characteristic of samples collected from two sites and

relative abundance of Serratia related sequences were assessed by the analysis of

similarity (ANOSIM) of the square root-transformed Bray-Curtis similarity data.

Differences were visualized with nonmetric multidimensional scaling (NMDS) plots

generated in R (R Development core team; http://www.R-project.org) using vegan

community ecology package. Association of different biogeochemical variables (Soil

texture, organic matter N, P, K, EC, pH and Serratia related sequences abundance) at

the different sites were assessed through the “envfit” function in the R software. Factors

showed significant association (P <0.01) were reported in NMDS plot.

2.10 Nucleotide Sequence Accession Numbers

16S rRNA gene sequences from rhizospheric soil and nodules of chickpea were

submitted in BioProject ID PRJNA340950 and nifH gene sequences from rhizospheric

soil and nodules of chickpea were submitted in BioProject ID PRJNA340949. Serratia

http://rdp.cme.msu.edu/


20

affiliated 16S rRNA gene sequences from chickpea root nodules were deposited in

GenBank under the accession numbers (KU299962 to KU300940). nifH gene

sequences from bacterial isolates were deposited in GenBank under the accession

numbers (LT604894 to LT604903). 16S rRNA gene sequences for bacterial isolates

were also submitted to GenBank under different accession numbers given in result

section Table 3.3.

21

3 Results

3.1 Isolation and Identification of Bacterial Isolates

3.1.1 Isolation and Identification of Bacteria from Rhizospheric Soil

and Root Nodules of Chickpea

In the present study, bacterial isolates were obtained from the rhizospheric soil and root

nodules of chickpea. Rhizospheric soil and nodule samples were collected from fields

under chickpea cultivation at 5 different locations i.e., Chowk Munda (District

Muzaffargarh), Kallar Syedan (District Rawalpindi), NIBGE, NIAB, AARI (District

Faisalabad), NIFA (District Peshawar) and Thal desert (Districts Bhakhar, Jhang and

Khushab) (Figure 3.1).

Figure 3-1 Sample collection from different chickpea growing area.

A= NIBGE Experimental Field, District Faisalabad, B= NIFA Experimental Fields,

District Peshawar, C= Kallar Syedan, District Rawalpindi, D= Chowk Munda,

District Muzaffargarh, E, F= Thal desert, District Bhakkar, G, H= Thal desert,

District Khushab and I= Thal desert, District Jhang

3. Results

22

Soil characterization was done (Table 3.1). The soil at Thal desert and Chowk Munda

were sandy loams, at NIFA and Kallar Syedan the soil were silty clay loams and at

NIBGE, NIAB and AARI, the soil were clay loams. The soil at NIBGE, NIAB, AARI,

NIFA and Kallar Syedan have more organic matter, N, P and K contents as compared

to the soils from Chowk Munda and Thal desert. Among the total 60 isolates obtained

in the present study, 6 isolates were purified from samples collected from Chowk

Munda area and 5 isolates were obtained from sample collected from Kallar Syedan

area. From the samples collected from NIBGE, NIFA and Thal desert, 21, 5 and 23

isolates were obtained, respectively. The isolates purified in the present study included

45 isolates from Desi-type and 15 isolates from Kabuli-type chickpea. Twenty-three

isolates originated from nodules of chickpea and the remaining majority of bacterial

isolates (37 isolates) were obtained from rhizospheric soil. Among root nodule

endophytes 2 isolates (isolates 5D and RTL100) were specifically targeted in an attempt

to purify Serratia strains on LB medium on the basis of colony morphology (reddish

pigment). The isolates included both the Gram positive (11 bacterial isolates) as well

as Gram negative (49 bacterial isolates) bacteria. Majority of the isolates were motile

Gram negative rods (Table 3.2; Figure 3.2).

Figure 3-2 Colony morphology of the isolates obtained from the root nodules

and rhizospheric soil of chickpea on LB and YMA media.

Serratia colonies on LB, Serratia sp. 5D, Serratia sp. RTL100, Microbacterium sp.

RTN145, Mesorhizobium sp. NTY7 and Ensifer sp. NFY8.

3. Results

23

Table 3.1 Physio-chemical characteristics of soil samples collected from

different localities

Parameter Thal desert

Chowk

Munda

NIFA Kallar

Syedan

NIBGE,

NIAB and

AARI

Latitudea 30°44'29.6"N

to

32°14'12.8"N

30° 35'

04.3" N

34° 00'

55.9"N

33° 22'

37.8" N

31° 23'

42.9" N

Longitudea 71°10'07.3"E

to

72°06'48.3"E

71° 14'

55.2" E

71° 42'

46.3"E

73° 30'

22.4" E

73° 01'

36.6" E

Altitudeb (m) 182 142 304 515 183

Annual

rainfallc (mm)

150-200 150-200 300-400 900-1000 350-450

Rainfall

during crop

seasonc (mm)

20-60 20-60 150-200 175-250 50-100

Date of

Sowing

October October November October November

Date of

Harvesting

April April April April April

Annual Tempc

(°C)

0-50 0-50 0-40 0-40 0-50

Temp during

crop seasonc

(°C)

0-40 0-40 -04-35 -04-35 0-40

Sandd (%) 71±2 65±3 4±3 19±2 41±1.5

Siltd (%) 20±1.5 20±1.5 64±2 55±3 30±1

Clayd (%) 9±1 15±1.5 32±2 26±1 29±1

Soil textured Sandy Loam Sandy Loam Silty clay

loam

Silty clay

loam

Clay loam

Organic

matterd (%)

0.294±0.024 0.294±0.024 1.4±0.21 0.6±0.02 0.6±0.065

pH d 8.06±0.093 8.64±0.13 7.7±0.51 7.7±0.25 8.1±0.057

ECd (dS/m) 0.396±0.022 0.396±0.022 0.65±0.05 0.31±0.05 0.402±0.025

Total Pd*

(µg/g)

559±5.3 599±8.56 1190±9.39 1258±15.9 1156±20.7

Available Pd* 3.118±0.25 3.118±0.25 1.74±0.025 1.84±0.15 7.92±0.6

Available Kd* 58.6±4.26 58.6±4.26 86.718±0.99

1

70±2.25 191.2±19.8

Available Nd 0.00414±0.00

2

0.0047±0.00

3

0.0075±0.02

1

0.0068±0.00

4

0.0087±0.04

0

Source a= Google Earth, b= Soil survey of Pakistan, c= Pakistan metrological

department, d= this study and d*= µg/g

3. Results

24

Table 3.2 Morphological characteristics of the bacterial isolates obtained

from rhizospheric soil and root nodules of chickpea

Sr.# /Isolate Isolation Source Location Colony and cell morphology

1. NFY135 Kabuli-type

rhizospheric soil

NIBGE Transparent, round, wrinkled colonies;

Cells motile, Gram -ve rods

2. NFN155 Desi-type

rhizospheric soil

NIBGE Transparent, round colonies; Cells

motile, Gram -ve rods

3. NFY133 Desi-type

rhizospheric soil

NIFA Slightly opaque, round colonies; Cells


4. JCN110 Kabuli-type

rhizospheric soil

NIBGE Greyish, round colonies; Cells motile,

Gram -ve rods

5. NTY29 Desi-type

rhizospheric soil

Thal

desert

Whitish, irregular colonies; Cells motile,

Gram +ve rods

6. NTY33 Desi-type

rhizospheric soil

Thal

desert


Gram +ve rods

7. RTY42 Desi-type

rhizospheric soil

Thal

desert


Gram +ve rods

8. JTN112 Desi-type

rhizospheric soil

Thal

desert


Gram +ve rods

9. JSN114 Kabuli-type

rhizospheric soil

NIBGE Whitish, irregular colonies; Cells motile,

Gram +ve rods

10. NTN143 Desi-type

rhizospheric soil

Thal

desert


Gram +ve rods

11. NFN149 Kabuli-type

rhizospheric soil

NIBGE Whitish, irregular colonies; Cells motile,

Gram +ve rods

12. JTN113 Desi-type

rhizospheric soil

Thal

desert

Whitish shiny, round colonies; Cells

non-motile, Gram –ve rods

13. NFY136 Desi-type

rhizospheric soil

Chowk

Munda



14. NTY140 Desi-type

rhizospheric soil

Thal

desert




rhizospheric soil

Kallar

Syedan

Whitish, round colonies; Cells motile,

Gram –ve rods


rhizospheric soil

Kallar

Syedan


Gram -ve rods

17. RTN142 Desi-type

rhizospheric soil

Thal

desert


Gram -ve rods

18. JCN109 Kabuli-type

rhizospheric soil

NIFA Yellowish, round colonies; Cells motile,

Gram -ve rods


nodule

NIBGE White gummy, round colonies; Cells


20. NFY124 Desi-type nodule Thal

desert

White gummy, round colonies; Cells


21. NTY34 Desi-type

rhizospheric soil

Chowk

Munda

Whitish, round colonies; Cells non-

motile, Gram –ve rods

22. NTY38 Desi-type

rhizospheric soil

Thal

desert

Whitish, round colonies; Cells non-


Cont…

3. Results

25

23. NTY48 Desi-type

rhizospheric soil

NIBGE Whitish, round colonies; Cells non-



rhizospheric soil



25. NTY36 Desi-type

rhizospheric soil




rhizospheric soil



27. NTY54 Desi-type

rhizospheric soil

Chowk

Munda

Orange, round colonies; Cells non-

motile, Gram +ve rods

28. RTL99 Desi-type

rhizospheric soil

Thal

desert

Orange, round colonies; Cells non-


29. NTY3 Desi-type nodule Chowk

Munda

White gummy, slightly irregular or round

colonies; Cells motile, Gram –ve rods

30. NPY4 Desi-type nodule NIFA White gummy, slightly irregular or round


31. NTY5 Kabuli-type

nodule

Thal

desert

(AZRI)




nodule

NIBGE White gummy, slightly irregular or round


33. NTY7 Desi-type nodule Thal

desert




desert



35. NRY10 Desi-type nodule Kallar

Syedan



36. NFY11 Desi-type nodule NIBGE White gummy, slightly irregular or round


37. NFY12 Desi-type nodule NIBGE White gummy, slightly irregular or round



nodule

NIBGE White gummy, slightly irregular or round


39. RTN145 Desi-type

rhizospheric soil

Thal

Desert

Light yellowish, round colonies; Cells



nodule

NIBGE Beige, round colonies; Cells motile,

Gram -ve rods

41. RTN154 Desi type nodule Thal

Desert

Beige, round colonies; Cells motile,

Gram -ve rods

42. NFN151 Desi-type nodule Chowk

Munda

Creamy, round colonies; Cells motile,

Gram +ve rods

43. RSY14 Desi-type

rhizospheric soil

Kallar

Syedan

Brownish, round colonies; Cells motile,

Gram -ve rods

44. NTY31 Desi-type

rhizospheric soil

NIBGE Brownish, round colonies; Cells motile,

Gram -ve rods


Desert


Gram -ve rods

Cont…

3. Results

26

46. RTY50 Desi-type

rhizospheric soil

Thal

Desert


Gram -ve rods

47. NTY51 Desi-type nodule NIBGE Brownish, round colonies; Cells motile,

Gram -ve rods


rhizospheric soil


Gram -ve rods


rhizospheric soil

Thal

Desert


Gram -ve rods


rhizospheric soil


Gram -ve rods


rhizospheric soil

Chowk

Munda


Gram -ve rods


rhizospheric soil

Thal

Desert


Gram -ve rods

53. NFN147 Kabuli-type

rhizospheric soil


Gram -ve rods

54. NTN153 Desi-type

rhizospheric soil

NIFA Brownish, round colonies; Cells motile,

Gram -ve rods


Desert


colonies; Cells motile, Gram -ve rods

56. JSN115 Desi-type nodule NIFA White gummy, slightly irregular or round


57. NTN152 Desi-type nodule Kallar

Syedan



58. 5D Desi-type nodule NIBGE Redish, round colonies; Cells motile,

Gram -ve rods

59. RTY59 Desi-type nodule Thal

Desert

Redish, round colonies; Cells motile,

Gram -ve short rods

60. RTL100 Desi-type nodule Thal

Desert

Redish, round colonies; Cells motile,

Gram -ve rods

3.1.2 Identification of Bacterial Isolates

For identification of the isolates, 16S rRNA gene was amplified from the genomic DNA

(Figure 3.3, 3.4) of all the bacterial isolates (60) obtained in the present study. 16S

rRNA gene sequence analysis of the majority of bacterial isolates showed more than 98

% similarity with the nucleotide sequence of closely related bacterial type strains (Table

3.3). However, 16S rRNA gene sequence of the bacterial isolate JSN115 showed 97.28

% similarity with Rhizobium massiliae 90AT (AF531767) and the isolate NFN155

showed 97.40 % similarity with Achromobacter xylosoxidans NBRC 15126T

(CP006958).

3. Results

27

Figure 3-3 Genomic DNA extracted from isolates obtained from root nodules

and rhizospheric soil of chickpea.

Lane 1: Ensifer sp. NFY8; Lane 2: Kocuria sp. RTL99; Lane 3:

Mesorhizobium sp. NTY7; Lane 4: Serratia sp. 5D; Lane 5: Serratia sp. RTL100;

Lane 6: Acinetobacter sp. NFY133; Lane 7: Pseudomonas sp. RSY14 and Lane 8:

Mesorhizobium sp. NTY3

In the present study 60 bacterial isolates were obtained from rhizospheric soil

and surface sterilized nodules of Desi and Kabuli-type of chickpea. 16S rRNA gene

sequence analysis revealed that the isolates represented five classes i.e., γ-

Proteobacteria (23 isolates), α-Proteobacteria (20 isolates), Firmibacteria (8

isolates), β-Proteobacteria (6 isolates) and Actinobacteria (3 isolates) and belonged to

18 different bacterial genera. Pseudomonas was the most abundant genus with 12

isolates, followed by Mesorhizobium (10 isolates), Bacillus (7 isolates), Enterobacter

(4 isolates), Bordetella (3 isolates), Bosea (3 isolates), Rhizobium (3 isolates), Serratia

(3 isolates), Achromobacter (2 isolates), Ensifer (2 isolates), Klebsiella (2 isolates),

Kocuria (2 isolates), Ochrobactrum (2 isolates), Acinetobacter (1 isolate), Aeromonas

(1 isolate), Duganella (1 isolate), Microbacterium (1 isolate) and Paenibacillus (1

isolate).

http://www.bacterio.net/gammaproteobacteria.html

http://www.bacterio.net/gammaproteobacteria.html

http://www.bacterio.net/alphaproteobacteria.html

http://www.bacterio.net/bacilli.html

http://www.bacterio.net/actinobacteria.html

3. Results

28

Phylogenetic analysis of α-proteobacterial isolates (Figure 3.5) indicated that

10 Mesorhizobium strains make clusters with Mesorhizobium robiniae CCNWYC 115T

(EU849582) and other closely related strain was Mesorhizobium muleiense CCBAU

83963T (HQ316710). Mesorhizobium mediterraneum UPM-Ca36T (L38825) and

Mesorhizobium temperatum SDW018T (AF508208) were also closely related strains to

mesorhizobial isolates obtained in the present study. In this phylogenetic tree, the two

Ensifer isolates formed cluster with Ensifer saheli ORS 609T (X68390) and Ensifer

mexicanus ITTG R7T (DQ411930) and the three Rhizobium isolates formed cluster with

Rhizobium pusense NRCPB10T (FJ969841). Both the Ochrobactrum isolates identified

in the present study clustered with Ochrobactrum pseudintermedium ADV31T

(DQ365921) in the phylogenetic tree. All the three Bosea isolates made cluster with

Bosea eneae 34614T (AF288300).

Phylogenetic analysis of β-proteobacterial isolates (Figure 3.6) showed that the

three strains of Bordetella sp. formed cluster with Bordetella petrii DSM 12804T

(AJ249861) and the isolates Duganella sp. clustered with Duganella violaceinigra YIM

31327T (AY376163).

Phylogenetic analysis of γ-proteobacterial isolates (Figure 3.7) indicated that a

group of five Pseudomonas isolates formed cluster with Pseudomonas plecoglossicida

NBRC 103162T (BBIV01000080). The remaining seven Pseudomonas isolates formed

a distinct cluster with Pseudomonas geniculata ATCC 19374T (AB021404.

Acinetobacter isolate formed the cluster with Acinetobacter pittii LMG 1035T

(HQ180184). Aeromonas isolate formed cluster with Aeromonas sobria ACC 43979T

(X74683). The Serratia sp. RTY59 formed cluster with Serratia nematodiphila DSM

21420T (JPUX01000001). The remaining two Serratia isolates formed the cluster with

Serratia marcescens subsp. marcescens DSM 30121T (AJ233431). Klebsiella isolates

formed cluster with Klebsiella pneumoniae subsp. ozaenae ATCC 11296T (Y17654).

Four isolates of Enterobactor sp. formed cluster with Enterobacter cloacae subsp.

cloacae ATCC 13047T (CP001918).

Phylogenetic analysis of actinobacterial isolates (Figure 3.8) indicated that

Kocuria sp. NTY54 and Kocuria sp. RTL99 formed cluster with Kocuria polaris CMS

76orT (AJ278868). Microbacterium sp. RTN145 formed cluster with Microbacterium

paraoxydans CF36T (AJ491806).

3. Results

29

Phylogenetic analysis of firmibacterial isolates (Figure 3.9) indicated that genus

Bacillus spp. was divided in three groups and formed three clusters with Bacillus

licheniformis ATCC 14580T (AE017333), Bacillus aerophilus 28KT (AJ831844) and

Bacillus safensis FO-36bT (ASJD01000027). Paenibacillus isolate NFN151 formed

cluster with Paenibacillus lautus NRRL NRS-666T (D78473).

Figure 3-4 PCR-amplification of 16S rRNA gene from bacterial isolates.

Lane 1: 1 kb ladder (Fermentas, Germany); Lane 2: Ensifer sp. NFY8; Lane

3: Mesorhizobium sp. NTY7; Lane 4: Serratia sp. 5D; Lane 5: negative control; Lane

6: 1 kb ladder (Fermentas, Germany)

3. Results

30

Figure 3-5 16S rRNA sequence-based phylogenetic tree of α-Proteobacteria

isolated from root nodules and rhizospheric soil of chickpea constructed by

Maximum Likelihood method.

Maximum likelihood bootstrap node support values ≥50. shown at the nodes. The

isolates (bold letters) identified in the present study were: a10 Mesorhizobium isolates

i.e., NTY3, NPY4, NTY5, NFY6, NTY7, NTY9, NRY10, NFY11, NFY12 and

NFY13. b2 Ensifer isolates i.e., NFY124 and NFY8. c3 Rhizobium isolates i.e.,

NTY40, JSN115 and NTN152. d2 Ochrobactrum isolates i.e., NFY131 and RTN154. e3 Bosea isolates i.e., NFY126, NFY130 and RTN142.

3. Results

31

Figure 3-6 16S rRNA sequence-based phylogenetic tree of β-Proteobacteria

isolated from the rhizospheric soil of chickpea constructed by maximum

likelihood method.

Only maximum likelihood bootstrap node support values ≥50 are shown at the nodes.

The isolates (bold letters) identified in the present study (accession # given in

brackets) were: a3 Bordetella isolates i.e., JTN113 (LK936576), NFY136 (LK936589)

and NTY140 (LK936591). a2 Achromobacter isolates i.e., NFY135 (LK936588) and

NFN155 (LK936601)

3. Results

32

Figure 3-7 16S rRNA sequence-based phylogenetic tree of γ-Proteobacteria

isolated from root nodule and rhizospheric soil of chickpea constructed by

maximum likelihood method.


The isolates (bold letters) identified in the present study were: a5 Pseudomonas

isolates i.e., NTY39, RTY50, NTY51, NTY123 and NFY134. b2 Serratia isolates i.e.,

5D and RTL100. c2 Klebsiella isolates i.e., NTY36 and NFY121. d4 Enterobacter

isolates i.e., NTY38, NTY34, NTY48 and NFY132. e7 Pseudomonas isolates i.e.,

RSY14, NTY31, NFY122, NFY125, NTY139, NFN147 and NTN153.

3. Results

33

Figure 3-8 16S rRNA sequence-based phylogenetic tree of Actinobacteria

isolated from rhizospheric soil of chickpea constructed by maximum likelihood

method.



brackets) were: a2 Kocuria isolates i.e., NTY54 (LK936570) and RTL99 (LK936572).

3. Results

34

Figure 3-9 16S rRNA sequence-based phylogenetic tree of Firmibacteria

isolated from root nodule and rhizospheric soil of chickpea constructed by




brackets) were: a2 Bacillus isolates i.e., JSN114 (LK936577) and NTN143

(LK936593). b4 Bacillus isolates i.e., NTY29 (LK936561), NTY33 (LK936563),

RTY42 (LK936567) and JTN112 (LK936575).

3. Results

35

Table 3.3 Identification of the bacterial isolates obtained from rhizospheric

soil and root nodules of chickpea on the basis of 16S rRNA gene sequence

analysis

Sr. No.

/Isolate

name

Isolates

identified

(Accession No.)

Closest type strain in EzTaxon database; %

sequence similarity

1. NFY135 Achromobacter

sp. (LK936588)

Achromobacter xylosoxidans NBRC 15126T

(CP006958); 99.88

2. NFN155 Achromobacter

sp. (LK936601)

Achromobacter xylosoxidans NBRC 15126T

(CP006958); 97.40

3. NFY133 Acinetobacter

sp. (LK936586)

Acinetobacter pittii CIP

70.29T (APQP01000001); 99.90

4. JCN110 Aeromonas sp.

(LK936574)

Aeromonas sobria ACC 43979T (X74683); 99.64

5. NTY29 Bacillus sp.

(LK936561)

Bacillus safensis FO-36bT (ASJD01000027); 100

6. NTY33 Bacillus sp.

(LK936563)

Bacillus safensis FO-36bT (ASJD01000027);

99.78

7. RTY42 Bacillus sp.

(LK936567)


8. JTN112 Bacillus sp.

(LK936575)


9. JSN114 Bacillus sp.

(LK936577)

Bacillus aerophilus 28KT (AJ831844); 99.39

10. NTN143 Bacillus sp.

(LK936593)

Bacillus aerophilus 28KT (AJ831844); 99.39

11. NFN149 Bacillus sp.

(LK936596)

Bacillus licheniformis ATCC 14580T

(AE017333); 97.92

12. JTN113 Bordetella sp.

(LK936576)

Bordetella petrii DSM 12804T (AM902716);

99.28

13. NFY136 Bordetella sp.

(LK936589)


99.14

14. NTY140 Bordetella sp.

(LK936591)


99.23

15. NFY126 Bosea sp.

(LK936583)

Bosea eneae 34614T (AF288300); 99.87

16. NFY130 Bosea sp.

(LK936584)

Bosea eneae 34614T (AF288300); 100

17. RTN142 Bosea sp.

(LK936592)

Bosea eneae 34614T (AF288300); 97.60

18. JCN109 Duganella sp.

(LK936573)

Duganella violaceinigra YIM

31327T (AY376163); 99.01

Cont…

3. Results

36

19. NFY8 Ensifer sp.

(LT604888)

Ensifer saheli LMG 7837T (X68390); 99.42

20. NFY124 Ensifer sp.

(LK936581)

Ensifer saheli LMG 7837T (X68390); 99.58

21. NTY34 Enterobacter

sp. (LK936564)

Enterobacter cloacae subsp. dissolvens LMG

2683T (Z96079); 99.89


sp. (HE995791)


2683T (Z96079); 99.90


sp. (LK936568)


2683T (Z96079); 99.89

24. NFY132 Enterobacter

sp. (LK936586)


2683T (Z96079); 99.78

25. NTY36 Klebsiella sp.

(LK936565)

Klebsiella variicola DSM 15968T (CP010523);

99.62

26. NFY121 Klebsiella sp.

(LK936579)

Klebsiella pneumoniae subsp. ozaenae ATCC

11296T (Y17654); 99.74

27. NTY54 Kocuria sp.

(LK936570)

Kocuria polaris CMS 76orT (JSUH01000031);

99.40

28. RTL99 Kocuria sp.

(LK936572)

Kocuria polaris CMS 76orT (JSUH01000031);

99.64

29. NTY3 Mesorhizobium

sp. (LT604883)

Mesorhizobium robiniae CCNWYC 115T

(EU849582); 100

30. NPY4 Mesorhizobium

sp. (LT604884)


(EU849582); 100


sp. (LT604885)


(EU849582); 99.85

32. NFY6 Mesorhizobium

sp. (LT604886)


(EU849582); 100


sp. (LT604887)


(EU849582); 100


sp. (LT604889)


(EU849582); 99.93

35. NRY10 Mesorhizobium

sp. (LT604890)


(EU849582); 100


sp. (LT604891)


(EU849582); 99.85


sp. (LT604892)


(EU849582); 100


sp. (LT604893)


(EU849582); 100

39. RTN145 Microbacterium

sp. (LK936594)

Microbacterium paraoxydans CF36T

(AJ491806); 97.95

Cont…

3. Results

37

40. NFY131 Ochrobactrum

sp. (LK936585)

Ochrobactrum pseudintermedium ADV31T

(DQ365921); 99.88

41. RTN154 Ochrobactrum

sp. (LK936600)

Ochrobactrum pseudintermedium ADV31T

(DQ365921); 97.87

42. NFN151 Paenibacillus

sp. (LK936597)

Paenibacillus lautus NRRL NRS-666T (D78473);

97.92

43. RSY14 Pseudomonas

sp. (LK936560)

Pseudomonas hibiscicola ATCC 19867T

(AB021405); 99.04

44. NTY31 Pseudomonas

sp. (LK936562)


(AB021405); 99.38


sp. (HE995792)

Pseudomonas taiwanensis BCRC 17751T

(EU103629); 99.62

46. RTY50 Pseudomonas

sp. (LK936569)

Pseudomonas monteilii NBRC 103158T

(BBIS01000088); 99.86


sp. (HE995795)

Pseudomonas taiwanensis BCRC 17751T

(EU103629); 99.91

48. NFY122 Pseudomonas

sp. (LK936580)


(AB021405); 99.40


sp. (HE995794)

Pseudomonas mosselii CIP 105259T (AF072688);

100


sp. (LK936582)


(AB021405); 99.40


sp. (HE995793)

Pseudomonas plecoglossicida NBRC 103162T

(BBIV01000080); 99.90


sp. (LK936590)


(AB021405); 99.84

53. NFN147 Pseudomonas

sp. (LK936595)

Pseudomonas geniculata ATCC 19374T

(AB021404); 99.90

54. NTN153 Pseudomonas

sp. (LK936599)


(AB021405); 97.63

55. NTY40 Rhizobium sp.

(LK936566)

Rhizobium pusense NRCPB10T (FJ969841); 100

56. JSN115 Rhizobium sp.

(LK936578)

Rhizobium massiliae 90AT (AF531767); 97.28

57. NTN152 Rhizobium sp.

(LK936598)

Rhizobium radiobacter ATCC 19358T

(AJ389904); 99.24

58. 5D Serratia

sp. (HE804807)

Serratia marcescens subsp. marcescens ATCC

13880T (JMPQ01000005); 99.81

59. RTY59 Serratia

sp. (LK936571)

Serratia nematodiphila DSM 21420T

(JPUX01000001); 99.43

60. RTL100 Serratia

sp. (HE995790)

Serratia marcescens subsp. marcescens ATCC

13880T (JMPQ01000005); 99.74

3. Results

38

3.1.3 Amplification of nifH Gene from Bacterial Isolates

Partial nifH gene was amplified using conserved primers from the bacterial

isolates showing colony and cell morphology as well as 16S rRNA sequence similar to

Mesorhizobium. PCR product of expected size (370 bp) was obtained from all the

Mesorhizobium strains tested in this study (Figure 3.10). Partial nifH PCR product from

10 Mesorhizobium isolates was sequenced and submitted to EMBL database. The

Accession # obtained were LT604894 to LT604903. The nifH sequence of all bacterial

isolates showed 99 % similarity with Mesorhizobium muleiense CCBAU 83963T

(HQ316767). Phylogenetic analysis of nifH gene (Figure 3.11) indicated that

Mesorhizobium spp. formed cluster with Mesorhizobium muleiense CCBAU 83963T

(HQ316767).

Figure 3-10 PCR amplification of partial nifH gene from Mesorhizobium

isolates obtained from root nodule of chickpea.

Lane 1, 1kb DNA marker (Thermo scientific, Germany); Lane 2, isolate NTY3; Lane

3, isolate NPY4; Lane 4, isolate NTY5; Lane 5, isolate NFY6; Lane 6, isolate NTY7;

Lane 7, negative control

3. Results

39

Figure 3-11 Nitrogenase reductase (nifH) sequence-based phylogenetic tree of

Mesorhizobium strains isolated from root nodule of chickpea constructed by



Sequences of isolates (bold letters) identified in the present study were identical and

have accession no LT604894 to LT604903.

3.2 Characterization of the Bacterial Isolates

3.2.1 Confirmation of Nodulation Ability of Endophytic Bacterial

Isolates

Plastic jar experiments were conducted to assess the nodulation ability of all endophytes

isolated from sterilized nodules of chickpea growing in different locations of Pakistan.

The seedlings were grown in sterilized sand. Ten bacterial isolates NTY3, NPY4,

NTY5, NFY6, NTY7, NTY9, NRY10, NFY11, NFY12 and NFY13 successfully

nodulated the chickpea seedlings and were therefore identified as Mesorhizobium

strains. Other 11 endophytic bacterial isolates failed to nodulate the host. All

Mesorhizobium strains resulted in increased dry weight of the host plants over

endophytic isolates and non-inoculated control (Table 3.4, 3.5; Figure 3.12, 3.13).

3. Results

40

Figure 3-12 Effect of Mesorhizobium spp. on growth and nodulation of

chickpea plants grown in sterilized sand.

Strains used: Mesorhizobium sp. NTY3 and Mesorhizobium sp. NTY7

Figure 3-13 Nodulation of chickpea by pure cultures of Mesorhizobium spp.

Strains used: Mesorhizobium sp. NTY3 and Mesorhizobium sp. NTY7 (Arrow:

nodules)

3. Results

41

Table 3.4 Nodulation of Desi-type chickpea by pure cultures of endophytes

Treatments No of

nodules

Dry weight

of nodules

(mg)

Dry weight of plant

(mg)

1. Non-inoculated control 0 0.00 661.33 ± 8.45 g

2. Mesorhizobium sp. NTY3 14 ± 1 21.00 ± 1.16 820.35 ± 32.05 ab

3. Mesorhizobium sp. NPY4 14 ± 1 20.02 ± 1.36 808.02 ± 24.89 ab


5. Mesorhizobium sp. NFY6 11 ± 1 16.64 ± 2.09 804.97 ± 17.96 ab

6. Mesorhizobium sp. NTY7 15 ± 2 21.00 ± 2.66 822.31 ± 33.58 a


8. Mesorhizobium sp. NRY10 12 ± 1 17.25 ± 1.68 809.81 ± 19.39 ab



11. Mesorhizobium sp. NFY13 11 ± 2 15.44 ± 3.17 804.84 ± 21.11 b

12. Ensifer sp. NFY8 0 0.00 739.28 ± 11.07 c

13. Ensifer sp. NFY124 0 0.00 724.13 ± 6.29 cd

14. Ochrobactrum sp. NFY131 0 0.00 700.46 ± 10.46 ef

15. Ochrobactrum sp. RTN154 0 0.00 720.27 ± 9.04 d

16. Paenibacillus sp. NFN151 0 0.00 722.81 ± 10.21 cd

17. Pseudomonas sp. NTY39 0 0.00 715.21 ± 9.58 de

18. Pseudomonas sp. NTY51 0 0.00 690.35 ± 11.53 f

19. Rhizobium sp. NTY40 0 0.00 710.02 ± 10.27 de

20. Rhizobium sp. JSN115 0 0.00 683.23 ± 11.74 f

21. Rhizobium sp. NTN152 0 0.00 698.62 ± 12.51 ef

22. Serratia sp. RTY59 0 0.00 656.30 ± 9.53 g

LSD at 0.05 17.39

3. Results

42

Table 3.5 Nodulation of Kabuli-type chickpea by pure cultures of

endophytes

Treatments No of

nodules

Dry weight of

nodules (mg)

Dry weight of plant

(mg)

1. Non-inoculated control 0 0.00 693.64 ± 11.97 gh


3. Mesorhizobium sp. NPY4 17 ± 2 27.36 ± 3.54 855.00 ± 28.02 ab


5. Mesorhizobium sp. NFY6 14 ± 3 23.29 ± 4.37 834.15 ± 26.04 b

6. Mesorhizobium sp. NTY7 17 ± 4 28.38 ± 6.34 860.10 ± 36.21 a


8. Mesorhizobium sp. NRY10 14 ± 3 22.01 ± 4.09 837.21 ± 27.85 b




12. Ensifer sp. NFY8 0 0.00 770.64 ± 11.97 c

13. Ensifer sp. NFY124 0 0.00 734.59 ± 21.47 de

14. Ochrobactrum sp. NFY131 0 0.00 730.00 ± 28.02 def

15. Ochrobactrum sp. RTN154 0 0.00 738.24 ± 20.33 d

16. Paenibacillus sp. NFN151 0 0.00 725.08 ± 21.78 def

17. Pseudomonas sp. NTY39 0 0.00 730.09 ± 20.23 def

18. Pseudomonas sp. NTY51 0 0.00 700.80 ± 13.07 gh

19. Rhizobium sp. NTY40 0 0.00 715.21 ± 30.27 efg

20. Rhizobium sp. JSN115 0 0.00 692.70 ± 13.65 h

21. Rhizobium sp. NTN152 0 0.00 711.62 ± 24.75 fgh

22. Serratia sp. RTY59 0 0.00 690.45 ± 18.64 h

LSD at 0.05 22.38

3.2.2 Indole-3-Acetic Acid (IAA) Production by the Bacterial Isolates

Bacterial isolates were investigated for the ability to produce indol-3-acetic acid (IAA)

in culture media containing tryptophan as precursor of IAA biosynthesis. Before

quantification of IAA on HPLC, qualitative test was performed which showed that 57

bacterial isolates produced colour (Figure 3.14). These isolates were further

investigated for quantification on HPLC. Among the isolates, Kocuria sp. RTL99

produced significantly higher amount of IAA (37.77 ± 1.20 µg/mL), followed by

Microbacterium sp. RTN145 (33.83 ± 1.07 µg/mL), Kocuria sp. NTY54 (33.23 ± 1.63

µg/mL), Ensifer sp. NFY8 (33.07 ± 0.90 µg/mL) and Mesorhizobium sp. NTY7 (30.83

± 0.70 µg/mL). Minimum IAA production was observed in Duganella sp. JCN109

(8.57 ± 0.51 µg/mL) (Table 3.6). Representative isolates (8 number) which produced

significantly higher amount of IAA, were further investigated for IAA production at

different temperatures. All tested strains showed the activity at 20 oC, 30 oC and 40 oC.

Maximum IAA was detected at 30 oC compared with 20 oC and 40 oC incubation

3. Results

43

temperatures. At 20 oC temperature all strains produced similar (non-significantly

different) amount of IAA. At 40 oC Ensifer sp. NFY8 produced significantly higher

amount of IAA (15.33 ± 2.05 µg/mL µg/mL), followed by Kocuria sp. RTL99 (14.83

± 1.55 µg/mL) (Table 3.7; Figure 3.15).

Figure 3-14 Qualitative test showing IAA production by different bacterial

isolates.

Strains used in this study: Ensifer sp. NFY8; Kocuria sp. RTL99; Mesorhizobium sp.

NTY7 and Microbacterium sp. RTN145.

3.2.3 Phosphate Solubilization by the Bacterial Isolates

Bacterial strains were grown on Pikovskaya medium agar plates to observe halo zone

formation as an indicator of P-solubilization activity. Halo zones were produced around

the colonies of 34 bacterial isolates grown for one weeks (Figure 3.16). Only these 34

isolates were selected for the quantification of P-solubilization. All the 34 bacterial

strains tested in the present study solubilized significant amount of TCP in pure culture

(Table 3.6). Maximum P-solubilization was observed in Serratia sp. 5D (119.94 ± 1.32

µg/mL), followed by Ensifer sp. NFY8 (114.28 ± 1.74 µg/mL) and Ensifer sp. NFY124

(107.27 ± 2.05 µg/mL).

The isolates which solubilized significantly higher amount of phosphate, were

further investigated for P-solubilization at different temperatures. All these bacterial

strains showed the solubilization activity at 20 oC, 30 oC and 40 oC. Maximum P was

solubilized at 30 oC compared with 20 oC and 40 oC incubation temperatures. At 20 oC

3. Results

44

and 40 oC temperature Serratia sp. 5D solubilized maximum P, followed by Ensifer sp.

NFY8 (Table 3.8; Figure 3.17).

Figure 3-15 IAA production by 8 selected strains at different incubation

temperatures.

Strains used in this study: Ensifer sp. NFY8 and NFY124; Kocuria sp. RTL99 and

NTY54; Mesorhizobium sp. NTY7; Microbacterium sp. RTN145 and Serratia sp. 5D

and RTL100.

Figure 3-16 Plate assay for phosphate solubilizing activity of bacterial isolates

Ensifer sp. NFY8 and Serratia sp. 5D.

3. Results

45

Figure 3-17 Phosphate solubilization by 7 selected strains at different

incubation temperatures.

Strains used in this study: Acinetobacter sp. NFY133; Bacillus sp. NTY33; Ensifer sp.

NFY8 and NFY124; Pseudomonas sp. RSY14 and Serratia sp. 5D and RTL100.

Table 3.6 Production of IAA* (µg/mL) and Phosphate solubilization**

(µg/mL) by bacterial strains in the growth medium.

Sr. No. / Isolate name IAA P-solubilization

1. Achromobacter sp. NFY135 17.23 ± 1.97 jk 28.50 ± 3.28 kl

2. Achromobacter sp. NFN155 15.07 ± 1.32 kl 19.63 ± 1.18 n

3. Acinetobacter sp. NFY133 11.80 ± 1.08 nopqrst 81.97 ± 1.76 e

4. Aeromonas sp. JCN110 9.60 ± 0.98 stuvw 12.03 ± 0.15 qrs

5. Bacillus sp. NTY29 12.37 ± 2.50 mnopq 14.60 ± 1.22 op

6. Bacillus sp. NTY33 12.37 ± 1.25 mnopq 80.83 ± 0.76 e

7. Bacillus sp. RTY42 12.40 ± 1.55 mnopq 26.67 ± 1.53 lm

8. Bacillus sp. JTN112 9.80 ± 1.04 rstuvw 31.97 ± 1.95 j

9. Bacillus sp. JSN114 10.10 ± 1.93 pqrstuvw 30.13 ± 1.00 jk

10. Bacillus sp. NTN143 11.73 ± 2.83 nopqrst 59.30 ± 1.13 h

11. Bacillus sp. NFN149 10.00 ± 2.65 pqrstuvw 54.70 ± 1.37 i

12. Bordetella sp. JTN113 9.67 ± 2.52 stuvw 12.63 ± 0.55 pqr

13. Bordetella sp. NFY136 10.30 ± 1.13 pqrstuvw 14.17 ± 0.31 opq

14. Bordetella sp. NTY140 11.43 ± 0.51 nopqrstu 15.50 ± 0.46 o

15. Bosea sp. NFY126 n.d. n.d.

16. Bosea sp. NFY130 n.d. n.d.

17. Bosea sp. RTN142 n.d. n.d.

18. Duganella sp. JCN109 8.57 ± 0.51 w n.d.

19. Ensifer sp. NFY8 33.07 ± 0.90 bc 114.28 ± 1.74 b

20. Ensifer sp. NFY124 28.67 ± 1.53 de 107.27 ± 2.05 c

21. Enterobacter sp. NTY34 10.00 ± 0.95 pqrstuvw n.d.

22. Enterobacter sp. NTY38 8.83 ± 0.96 vw n.d.

23. Enterobacter sp. NTY48 8.83 ± 1.76 vw n.d.

Cont…

3. Results

46

24. Enterobacter sp. NFY132 9.50 ± 1.50 stuvw n.d.

25. Klebsiella sp. NTY36 13.47 ± 1.31 lmn n.d.

26. Klebsiella sp. NFY121 12.83 ± 1.61 lmno n.d.

27. Kocuria sp. NTY54 33.23 ± 1.63 bc n.d.

28. Kocuria sp. RTL99 37.77 ± 1.20 a n.d.

29. Mesorhizobium sp. NTY3 28.00 ± 1.00 ef n.d.

30. Mesorhizobium sp. NPY4 26.00 ± 1.00 fg n.d.

31. Mesorhizobium sp. NTY5 25.00 ± 1.00 g n.d.

32. Mesorhizobium sp. NFY6 25.33 ± 2.08 g n.d.

33. Mesorhizobium sp. NTY7 30.83 ± 0.70 cd n.d.

34. Mesorhizobium sp. NTY9 28.83 ± 1.04 de n.d.

35. Mesorhizobium sp. NRY10 26.67 ± 3.51 efg n.d.

36. Mesorhizobium sp. NFY11 29.00 ± 2.00 de n.d.

37. Mesorhizobium sp. NFY12 26.10 ± 1.93 fg n.d.

38. Mesorhizobium sp. NFY13 24.63 ± 2.28 g n.d.

39. Microbacterium sp. RTN145 33.83 ± 1.07 b n.d.

40. Ochrobactrum sp. NFY131 14.80 ± 0.26 klm n.d.

41. Ochrobactrum sp. RTN154 13.63 ± 1.18 lmn n.d.

42. Paenibacillus sp. NFN151 10.63 ± 0.55 opqrstuvw n.d.

43. Pseudomonas sp. RSY14 12.30 ± 2.04 mnopqr 90.72 ± 0.75 d

44. Pseudomonas sp. NTY31 9.33 ± 2.52 tuvw 70.50 ± 0.50 f

45. Pseudomonas sp. NTY39 9.67 ± 1.53 stuvw 55.07 ± 0.80 i

46. Pseudomonas sp. RTY50 12.00 ± 3.00 nopqrs 14.97 ± 1.00 o

47. Pseudomonas sp. NTY51 11.13 ± 0.32 nopqrstuv 9.63 ± 1.52 tu

48. Pseudomonas sp. NFY122 9.97 ± 1.05 qrstuvw 10.80 ± 1.08 rst

49. Pseudomonas sp. NTY123 9.00 ± 1.00 uvw 13.97 ± 1.76 opq

50. Pseudomonas sp. NFY125 12.00 ± 1.00 nopqrs 26.53 ± 1.46 lm

51. Pseudomonas sp. NFY134 12.50 ± 0.50 mnop 31.27 ± 1.74 j

52. Pseudomonas sp. NTY139 9.40 ± 0.46 tuvw 21.10 ± 0.72 n

53. Pseudomonas sp. NFN147 9.20 ± 1.14 uvw 20.10 ± 1.25 n

54. Pseudomonas sp. NTN153 11.30 ± 1.57 nopqrstuv 68.27 ± 1.65 g

55. Rhizobium sp. NTY40 26.10 ± 1.01 fg 10.03 ± 2.47 stu

56. Rhizobium sp. JSN115 22.00 ± 1.73 h 8.33 ± 0.51 u

57. Rhizobium sp. NTN152 21.00 ± 1.00 hi 8.73 ± 1.62 tu

58. Serratia sp. 5D 19.23 ± 0.70 ij 119.94 ± 1.32 a

59. Serratia sp. RTY59 15.27 ± 0.93 kl 25.33 ± 1.36 m

60. Serratia sp. RTL100 18.50 ± 0.90 ij 30.60 ± 1.18 jk

LSD at 0.05 2.5076 2.2135

*Bacterial cultures were grown for 7 days in LB/TY medium containing tryptophan as

precursor of IAA. The values given are an average of 3 replicates.

**Bacterial cultures were grown for 15 days in Pikovskaya growth medium (pH 7)

containing insoluble tri-calcium phosphate. The values given are an average of 3

replicates. N.D. = Not determined

3. Results

47

Table 3.7 Production of IAA* by bacterial strains at different temperatures

Sr. No. / Strains Name IAA production (µg/mL)

20 oC 30 oC 40 oC

1. Ensifer sp. NFY8 11.00 ± 1.63 a 33.07 ± 0.90 b 15.33 ± 2.05 a

2. Ensifer sp. NFY124 9.33 ± 1.25 a 28.67 ± 1.53 d 12.67 ± 1.70 abc

3. Kocuria sp. NTY54 8.67 ± 1.24 a 33.23 ± 1.63 b 13.50 ± 0.41 ab

4. Kocuria sp. RTL99 10.33 ± 2.05 a 37.77 ± 1.20 a 14.83 ± 1.55 a

5. Mesorhizobium sp. NTY7 11.00 ± 1.63 a 30.83 ± 0.70 c 10.33 ± 1.25 cd

6. Microbacterium sp. RTN145 11.33 ± 2.62 a 33.83 ± 1.07 b 11.67 ± 1.69 bcd

7. Serratia sp. 5D 9.50 ± 0.41 a 19.23 ± 0.70 e 10.20 ± 0.73 cd

8. Serratia sp. RTL100 8.92 ± 0.29 a 18.50 ± 0.90 e 9.53 ± 0.94 d

LSD at 0.05 3.34 1.87 2.95

*Bacterial cultures were grown for 7 days in LB/TY medium containing tryptophan as

precursor of IAA. The values given are an average of 3 replicates.

Table 3.8 Phosphate solubilization* by bacterial strains at different

temperatures

Sr. No. /Strains Name P-solubilization (µg/mL)

20 oC 30 oC 40 oC

1. Acinetobacter sp. NFY133 7.33 ± 2.05 d 81.97 ± 1.76 e 5.67 ± 1.70 d

2. Bacillus sp. NTY33 24.00 ± 4.32 c 80.83 ± 0.76 e 48.33 ± 2.49 bc

3. Ensifer sp. NFY8 44.33 ± 6.65 ab 114.28 ± 1.74 b 57.33 ± 2.05 ab

4. Ensifer sp. NFY124 41.67 ± 6.24 b 107.27 ± 2.05 c 51.67 ± 8.50 bc

5. Pseudomonas sp. RSY14 23.33 ± 1.25 c 90.72 ± 0.75 d 42.67 ± 6.13 c

6. Serratia sp. 5D 50.54 ± 0.81 a 119.94 ± 1.32 a 60.94 ± 0.82 a

7. Serratia sp. RTL100 10.44 ± 0.36 d 30.60 ± 1.18 f 10.60 ± 1.31 d

LSD at 0.05 8.44 2.28 9.08

*Bacterial cultures were grown for 15 days in Pikovskaya growth medium (pH 7)

containing insoluble tri-calcium phosphate. The values given are an average of 3

replicates.

3.2.4 Organic Acid Production

Bacterial isolates which solubilized significantly higher amount of P at 20 oC, 30 oC

and 40 oC temperature, were further investigated for production of organic acids such

acetic, citric, gluconic, malic, succinic, lactic and oxalic acid. All strains produced the

acetic, citric, gluconic, and succinic acid (Table 3.9; Figure 3.18). Among the tested

strains Bacillus sp. NTY33 produced all 7 organic acids. Ensifer sp. produced more

amount of acetic, gluconic and lactic acid. Bacillus sp. NTY33 produced more amount

of citric and succinic acid among the tested strains.

3. Results

48

Figure 3-18 Organic acid production by bacterial strains.

Bacterial cultures were grown for 15 days at 30 oC in Pikovskaya growth medium (pH

7) containing insoluble tri-calcium phosphate. The values given are an average of 3

replicates.

Table 3.9 Organic acid production* (µg/mL) by bacterial strains in

Pikovskaya growth medium

Strains

Name

Acetic

Acid

Citric

Acid)

Gluconic

Acid

Malic

Acid

Succinic

Acid

Lactic

Acid

Oxalic

Acid

Bacillus sp.

NTY33

23.30 ±

1.06 b

5.20 ±

0.16 a

0.90 ±

0.24 b

0.70 ±

0.08 b

1.20 ±

0.16 a

1.00 ±

0.08 c

0.50 ±

0.16 a

Ensifer sp.

NFY8

27.50 ±

1.22 a

2.10 ±

0.22 c

2.20 ±

0.29 a

n.d. 1.05 ±

0.04 a

1.90 ±

0.14 b

n.d.

Ensifer sp.

NFY124

27.00 ±

1.63 a

2.40 ±

0.29 c

1.90 ±

0.08 a

n.d. 1.06 ±

0.04 a

1.53 ±

0.29 b

n.d.

Pseudomonas

sp. RSY14

23.47 ±

0.38 b

2.30 ±

0.08 c

0.52 ±

0.38 b

0.053 ±

0.04 c

0.50 ±

0.29 b

0.09 ±

0.01 d

n.d.

Serratia

sp. 5D

28.43 ±

0.42 a

3.10 ±

0.08 b

0.67 ±

0.42 b

1.07 ±

0.05 a

0.40 ±

0.22 b

3.45 ±

0.20 a

n.d.

Serratia

sp. RTL100

26.67 ±

0.94 a

3.07 ±

0.11 b

0.67 ±

0.26 b

1.00 ±

0.24 ab

0.37 ±

0.17 b

n.d. 0.70 ±

0.24 a

LSD at 0.05 2.27 0.38 0.66 0.31 0.39 0.39 0.58

*Bacterial cultures were grown for 15 days at 30 oC in Pikovskaya growth medium (pH

7) containing insoluble tri-calcium phosphate. The values given are an average of 3.

N.D. = Not detected

3. Results

49

3.3 Effect of Bacterial Inoculation on Chickpea

3.3.1 Effect of Bacterial Inocula on Chickpea Plants Grown in

Earthen Pots (Year 2012-13)

Earthen pot experiments were conducted to evaluate the effect of 10 selected bacterial

strains (efficient IAA producers and P-solubilizes) as single strain inoculants for

chickpea (Figure 3.19). Earthen pots were filled with soil collected from NIBGE

experimental fields. Two chickpea varieties, i.e., Punjab 2008 (Desi-type) and Noor

2009 (Kabuli-type) were used as host plants for the inoculation studies. The comparison

of the overall effect of inoculation on tested varieties revealed that seven stains

increased the number of nodules, dry weight of nodules, plant yield and plant straw

weight over non-inoculated control. Maximum grain yields obtained were 2.26, 2.25,

2.22 and 2.15 g/plant recorded in the treatments inoculated with Mesorhizobium sp.

NTY7, Ensifer sp. NFY8, Serratia sp. 5D, Kocuria sp. RTL99, respectively. The yield

were significantly higher than that of the non-inoculated control (i.e., 1.60 g/ plant).

Three strains (Bacillus sp. NTY33, Microbacterium sp. RTN145 and Pseudomonas sp.

RSY14) did not perform well as compared to non-inoculated control in all parameters

i.e, number of nodules, dry weight of nodules, plant yield and plant straw weight. Four

strains (Ensifer sp. NFY8, Kocuria sp. RTL99, Mesorhizobium sp. NTY7 and Serratia

sp. 5D) performed well as compared to other strains and non-inoculated control in all

the parameters (Table 3.10 and 3.11).


earthen pots. Strain used: Serratia sp. 5D. (Year 2012-13)

3. Results

50

Among the chickpea varieties tested, Desi-type gave significantly higher yield

in general (2.06 g grain/plant) than Kabuli-type (1.60 g grain/plant), which was true for

inoculated as well as non-inoculated treatments. Straw weight and number of nodules

were not significantly different in both types of chickpea. But in case of dry weight of

nodules, Kabuli-type have significantly higher dry weight of nodules in general (154.91

mg dry weight of nodules/plant) than Desi-type (116.73 mg dry weight of

nodules/plant), which was true for inoculated as well as non-inoculated treatments

(Table 3.10 and 3.11).

Table 3.10 Effect of bacterial inocula on number of nodules and dry weight of

nodules (mg/plant) of chickpea plant grown in earthen pots. (Year 2012-13)

T Desi-Type Chickpea Kabuli-Type Chickpea Overall effect of

treatments

NN DNN NN DNN NN DNN

T1 7.40 ab 111.00 e 7.40 ab 148.00 bc 7.40 B 129.50 B

T2 7.40 ab 111.00 e 7.40 ab 148.00 bc 7.40 B 129.50 B

T3 8.00 ab 120.00 de 7.80 ab 156.00 ab 7.90 AB 138.00 AB

T4 7.80 ab 117.00 e 7.80 ab 156.00 ab 7.80 AB 136.50 AB

T5 7.80 ab 117.00 e 7.80 ab 156.00 ab 7.80 AB 136.50 AB

T6 8.00 ab 120.00 de 8.00 ab 160.00 ab 8.00 AB 140.00 AB

T7 8.60 ab 129.00 cde 8.80 a 176.00 a 8.70 A 152.50 A

T8 7.20 b 108.00 e 7.20 b 144.00 bcd 7.20 B 126.00 B

T9 7.40 ab 111.00 e 7.60 ab 152.00 abc 7.50 B 131.50 B

T10 8.00 ab 120.00 de 7.80 ab 156.00 ab 7.90 AB 138.00 AB

T11 8.00 ab 120.00 de 7.60 ab 152.00 abc 7.80 AB 136.00 AB

7.78 A 116.73 B 7.75 A 154.91 A

T: Treatments; T1: Control; T2: Bacillus sp. NTY33; T3: Ensifer sp. NFY8; T4:

Ensifer sp. NFY124; T5: Kocuria sp. NTY54; T6: Kocuria sp. RTL99; T7:

Mesorhizobium sp. NTY7; T8: Microbacterium sp. RTN145; T9: Pseudomonas sp.

RSY14; T10: Serratia sp. 5D; T11: Serratia sp. RTL100; NN: Number of nodules per

plant and DNN: Dry weight of nodules per plant

Values are an average of 5 replicates. Different small letters in the same column

represent statistically different values and the capital letters represent overall effect of

multiple factors like bacterial inoculation and plant type.

LSD @ 0.05α for number of nodules X type of chickpea= 0.4233, LSD @ 0.05α for

number of nodules X Treatments = 0.9928, LSD @ 0.05α for number of nodules X type

of chickpea X Treatments = 1.4040, LSD @ 0.05α for dry weight of nodules X type of

chickpea= 7.6884, LSD @ 0.05α for dry weight of nodules X Treatments = 18.031 and

LSD @ 0.05α for dry weight of nodules X type of chickpea X Treatments = 25.500,

3. Results

51

Table 3.11 Effect of bacterial isolates on grain and straw yield (g/plant) of

chickpea grown in earthen pots. (Year 2012-13)

T Desi-Type

Chickpea

Kabuli-Type

Chickpea

Overall effect of

treatments

Grain

yield

Straw

yield

Grain

yield

Straw

yield

Grain

yield

Straw

yield

T1 1.82 fg 2.92 bc 1.37 h 2.91 c 1.60 C 2.91 C

T2 1.86 ef 2.92 bc 1.41 h 2.92 bc 1.63 C 2.92 C

T3 2.50 a 3.15 a 2.00 cd 3.19 a 2.25 A 3.17 A

T4 1.83 fg 2.96 bc 1.41 h 2.95 bc 1.62 C 2.95 BC

T5 1.78 g 2.93 bc 1.38 h 2.95 bc 1.58 C 2.94 BC

T6 2.36 b 2.96 bc 1.93 de 2.97 bc 2.15 B 2.96 BC

T7 2.50 a 3.23 a 2.02 c 3.22 a 2.26 A 3.22 A

T8 1.85 fg 2.93 bc 1.38 h 2.93 bc 1.62 C 2.93 C

T9 1.84 fg 2.92 bc 1.39 h 2.90 c 1.61 C 2.91 C

T10 2.47 a 3.02 b 1.97 cd 3.01 bc 2.22 A 3.01 B

T11 1.86 ef 2.94 bc 1.38 h 2.97 bc 1.62 C 2.96 BC

2.06 A 2.99 A 1.60 B 2.99 A

T: Treatments; T1: Control; T2: Bacillus sp. NTY33; T3: Ensifer sp. NFY8; T4: Ensifer

sp. NFY124; T5: Kocuria sp. NTY54; T6: Kocuria sp. RTL99; T7: Mesorhizobium sp.

NTY7; T8: Microbacterium sp. RTN145; T9: Pseudomonas sp. RSY14; T10: Serratia

sp. 5D and T11: Serratia sp. RTL100




LSD @ 0.05α for yield X type of chickpea= 0.0236, LSD @ 0.05α for yield X

Treatments = 0.0554, LSD @ 0.05α for yield X type of chickpea X Treatments =

0.0784, LSD @ 0.05α for straw X type of chickpea= 0.0327, LSD @ 0.05α for straw X

Treatments = 0.0767 and LSD @ 0.05α for straw X type of chickpea X Treatments =

0.1084

3. Results

52

3.3.2 Plant Growth Promoting Effect of Serratia spp. on Chickpea

Grown in Field (Year 2013-14)

Field experiments were conducted at NIBGE (District Faisalabad) and Thal desert

(District Khushab) to evaluate the effect of Serratia strains 5D and RTL100 as

inoculants for chickpea (Figure 3.20). Soil analysis indicated that the soil at the Thal

desert (District Khushab) was nutrient-deficient compared with the NIBGE soil

(District Faisalabad) (Table 3.12). Two chickpea varieties, i.e., Punjab 2008 (Desi-type)

and Noor 2009 (Kabuli-type) were used as host plants for inoculation studies at both

the experimental sites. The results revealed that inoculation with Serratia spp.

significantly increased the grain and straw yield of the tested crop varieties compared

to non-inoculated control at both localities (Table 3.13 and 3.14; Figure 3.21 and 3.22).

The comparison of the overall effect of inoculation on tested varieties on both sites

indicated that the maximum increase in grain yield was 1625.2 and 1496.2 kg/ha

recorded in the treatment inoculated with Serratia strain 5D and Serratia strain

RTL100, respectively which was significantly higher than that of the non-inoculated

control (i.e., 1339.7 kg/ha). Among the two strains, Serratia sp. 5D was the most

effective inoculant, causing up to 21.34 % and 18.60 % increase in grain and straw

yield, respectively over non-inoculated control (Table 13 and 14; Figure 19 and 20).

Among the chickpea varieties tested, Desi-type gave significantly higher yield in

general (1742.2 and 2870.8 kg/ha grain and straw, respectively) than Kabuli-type

(1232.1 and 2589.4 kg/ha grain and straw, respectively), which was true for inoculated

as well as non-inoculated treatments. Between the two sites used for cultivation of

chickpea, more yield of grain and straw was obtained at NIBGE (District Faisalabad)

with both varieties as compared to that of Thal desert (District Khushab). Irrespective

of chickpea variety, experimental area and the bacterial inoculants, the seed and straw

yield was found to be higher in all treatments supplemented with fertilizer as compared

to non-fertilized treatments (Table 3.13, 3.14; Figure 3.21, 3.22).

3.3.3 Plant Growth Promoting Effect of bacterial inocula on chickpea

grown in earthen pots (Year 2013-14)

Earthen pot experiments were conducted to evaluate the effect of 4 bacterial strains

(Ensifer sp. NFY8, Kocuria sp. RTL99, Mesorhizobium sp. NTY7 and Serratia sp. 5D)

as single strain inocula (Figure 3.23) as well as co-inoculation of Mesorhizobium sp.

NTY7 with Ensifer sp. NFY8, Kocuria sp. RTL99 and Serratia sp. 5D. The comparison

3. Results

53

of the overall effect of inoculation on tested varieties revealed that all strains (as single-

strain or co-inoculants) increased the number of nodules, dry weight of nodules, plant

yield and plant straw weight over non-inoculated control (Table 3.15, 3.16). Maximum

grain yield (2.35 g/plant) was recorded in the treatment inoculated with consortia of

Mesorhizobium sp. NTY7 and Ensifer sp. NFY8 which was significantly higher than

that of the non-inoculated control (i.e., 1.59 g/plant). Among the chickpea varieties

tested, Desi-type gave significantly higher yield in general (2.42 g grain /plant) than

Kabuli-type (1.94 g grain/plant), which was true for inoculated as well as non-

inoculated treatments. Among both types of chickpea, there as no significant difference

between straw weight and number of nodules. However, Kabuli-type showed

significantly higher dry weight of nodules in general (166.00 mg/plant) than Desi-type

(123.75 mg/plant), which was true for inoculated as well as non-inoculated treatments

(Table 3.15, 3.16).

Figure 3-20 Effect of Serratia sp. on growth of chickpea plants grown in field at

two different localities. (Year 2013-14)

3. Results

54

Figure 3-21 Effect of bacterial inoculation (Serratia spp.) on grain yield (kg/ha)

of chickpea

grown in fertile, irrigated area (NIBGE, District Faisalabad) and nutrient-deficient,

rainfed area (Thal, District Khushab). A= Desi-type full fertilizer; B= Desi-type no

fertilizer; C= Kabuli-type full fertilizer and D= Kabuli-type no fertilizer. (Detailed

statistical analysis is given in Table 3.13). Values are an average of 5 replicates. Error

bars represent the standard deviations (SD). (Year 2013-14)

3. Results

55

Figure 3-22 Effect of bacterial inoculation (Serratia spp.) on straw weight

(kg/ha) of chickpea

grown in fertile, irrigated area (NIBGE, District Faisalabad) and nutrient-deficient,

rainfed area (Thal, District Khushab). A= Desi-type full fertilizer; B= Desi-type no

fertilizer; C= Kabuli-type full fertilizer and D= Kabuli-type no fertilizer. (Detailed

statistical analysis is given in Table 3.14). Values are average of 5 replicates. Error

bars represent the standard deviations (SD). (Year 2013-14)

3. Results

56


Earthen pots.

Strains used: Mesorhizobium sp. NTY7, Ensifer sp. NFY8 and Serratia sp. 5D. (Year

2013-14)

Table 3.12 Characteristics of soil samples collected at the time of sowing

from fertile field of irrigated areas (NIBGE, District Faisalabad) and nutrient-

deficient field of rainfed areas (Thal desert, District Khushab). (Year 2013-14)

Parameter Thal Desert, District

Khushab

NIBGE, District

Faisalabad

Rainfall during crop

seasona (mm)

50 70

Soil texture Sandy Loam Clay loam

Organic matter (%) 0.294 ± 0.02 0.6 ± 0.06

pH 8.06 ± 0.09 8.1 ± 0.06

EC (dS/m) 0.40 ± 0.02 0.402 ± 0.02

Total P (µg/g soil) 559 ± 5 1156 ± 21

Available P (µg/g soil) 3.12 ± 0.25 7.9 ± 0.6

Available K (µg/g soil) 58.6 ± 4.26 191.2 ± 19.8

Available N (%) 0.004 ± 0.003 0.009 ± 0.040

Source a= Pakistan metrological department. All other information are from current

study.

3. Results

57

Table 3.13 Effect of bacterial inoculation (Serratia spp.) on grain yield (kg/ha)

of chickpea grown in fertile, irrigated area (NIBGE, District Faisalabad) and

nutrient-deficient, rainfed area (Thal desert, District Khushab). (Year 2013-

2014)

T Desi-Type Chickpea Kabuli-Type Chickpea

NIBGE,

District

Faisalabad

Thal, District

Khushab

NIBGE,

District

Faisalabad

Thal, District

Khushab

F NF F NF F NF F NF

Control 1843.2

c

1660.9

d

1514.6

e

1308.0

gh

1300.0

h

1148.7

ij

1047.1

k

895.4 l 1339.7

C

Serratia

sp. 5D

2119.2

a

1997.3

b

1793.2

c

1664.4

d

1528.4

e

1442.2

ef

1288.3

h

1171.6 i 1625.6

A

Serratia

sp.

RTL100

1974.5

b

1857.2

c

1655.3

d

1519.0

e

1403.3

fg

1330.2

gh

1172.0 i 1058.2

jk

1496.2

B

1979.0

A

1838.5

B

1654.4

C

1497.1

D

1410.5

E

1307.0

F

1169.1

G

1041.7

H

1908.7 A 1575.7 B 1358.8 C 1105.4 D

1742.2 A 1232.1 B

T: Treatment; F: Fertilized; NF: Non-fertilized; CT: Chickpea type (Desi or Kabuli);

Control: non-inoculated.

Different small letters in the same column represent statistically different values and

the capital letters represent overall effect of multiple factors like bacterial inoculation,

fertilizer application, plant type, experimental site.

LSD @ 0.05α for T X CT X F X L=101.63, LSD @ 0.05α for CT X F X L=58.676,

LSD @ 0.05α for CT X L=41.49, LSD @ 0.05α for T= 35.932 and LSD @ 0.05α for

CT= 29.338.

3. Results

58

Table 3.14 Effect of bacterial inoculation (Serratia spp.) on straw yield (kg/ha)

of chickpea grown in fertile, irrigated area (NIBGE, District Faisalabad) and

nutrient-deficient, rainfed area (Thal desert, District Khushab). (Year 2013-

2014)

T Desi-Type Chickpea Kabuli-Type Chickpea

NIBGE,

District

Faisalabad

Thal, District

Khushab

NIBGE,

District

Faisalabad

Thal, District

Khushab

F NF F NF F NF F NF

Control 3114.7

de

2489.4

l

2629.2

jkl

2233.2

mn

2828.3

gh

2307.8

m

2334.5

m

1746.4

0

2462.9

C

Serratia

sp. 5D

3508.7

a

2891.2

fg

3090.1

de

2746.9

ghijk

3294.1

bc

2800.6

ghi

2753.2

ghij

2282.4

mn

2920.9

A

Serratia

sp.

RTL100

3401.9

ab

2760.3

ghij

2985.0

ef

2598.6 kl 3192.9

cd

2710.5

hijk

2653.8

ijk

2148.8

n

2806.5

B

3341.8

A

2713.6

D

2901.4

C

2526.2

E

3105.1

B

2606.3

E

2580.5

E

2065.9

F

3027.7 A 2713.8 C 2855.7 B 2323.2 D

2870.8 A 2589.4 B

T: Treatment; F: Fertilized; NF: Non-fertilized; CT: Chickpea type (Desi or Kabuli);

Control: non-inoculated.

Different small letters in the same column represent statistically different values and

the capital letters represent overall effect of multiple factors like bacterial inoculation,

fertilizer application, plant type, experimental site.

LSD @ 0.05α for T X CT X F X L=148.55, LSD @ 0.05α for CT X F X L=85.765,

LSD @ 0.05α for CT X L=60.645, LSD @ 0.05α for T= 52.520 and LSD @ 0.05α for

CT=42.883.

3. Results

59

Table 3.15 Effect of bacterial isolates as single-strain inocula and as co-

inoculants on chickpea grown in earthen pots. (Year 2013-14)


treatments

No. of

nodules per

plant

Dry weight

of nodules

per plant

No. of

nodules per

plant

Dry weight

of nodules

per plant

No. of

nodules

per plant

Dry weight

of nodules

per plant

T1 7.00 e 105.00 h 7.00 e 140.00 def 7.00 D 122.50 D

T2 8.20 abcde 123.00 fgh 8.20 abcde 164.00 abcd 8.20 BC 143.50 BC

T3 8.00 abcde 120.00 fgh 7.80 bcde 156.00 bcde 7.90 BCD 138.00 BCD

T4 8.40 abcde 126.00 fgh 8.60 abcd 172.00 abc 8.50 ABC 149.00 ABC

T5 7.40 de 111.00 gh 7.60 cde 152.00 cde 7.50 CD 131.50 CD

T6 9.40 a 141.00 def 9.20 ab 184.00 a 9.30 A 162.50 A

T7 8.80 abcd 132.00 efg 9.00 abc 180.00 ab 8.90 AB 156.00 AB

T8 8.80 abcd 132.00 efg 9.00 abc 180.00 ab 8.90 AB 156.00 AB

8.25 A 123.75 B 8.30 A 166.00 A

T: Treatments; T1: Control; T2: Ensifer sp. NFY8; T3: Kocuria sp. RTL99; T4:

Mesorhizobium sp. NTY7; T5: Serratia sp. 5D; T6: Mesorhizobium sp. NTY7 + Ensifer

sp. NFY8; T7: Mesorhizobium sp. NTY7+ Kocuria sp. RTL99 and T8: Mesorhizobium

sp. NTY7+ Serratia sp. 5D








LSD @ 0.05α for dry weight of nodules X type of chickpea X Treatments = 25.833,

3. Results

60

Table 3.16 Effect of bacterial isolates as single strain inocula and co-

inoculation on grain and straw yield (g/plant) of chickpea grown in earthen pots.

(Year 2013-14)


treatments Grain

yield

Straw

yield

Grain

yield

Straw yield Grain

yield

Straw

yield

T1 1.80 i 2.92 g 1.37 j 2.92 g 1.59 E 2.92 E

T2 2.50 bc 3.12 f 2.00 fgh 3.14 ef 2.25 C 3.13 C

T3 2.36 d 2.96 g 1.93 h 2.96 g 2.15 D 2.96 DE

T4 2.50 bc 3.20 def 2.01 fg 3.18 def 2.26 C 3.19 BC

T5 2.47 c 3.01 g 1.97 gh 3.00 g 2.22 C 3.00 D

T6 2.60 a 3.36 ab 2.11 e 3.40 a 2.35 A 3.38 A

T7 2.52 bc 3.24 cde 2.03 fg 3.27 bcd 2.27 BC 3.25 B

T8 2.57 ab 3.32 abc 2.07 ef 3.35 abc 2.32 AB 3.34 A

2.42 A 3.14 A 1.94 B 3.15 A

T: Treatments; T1: Control; T2: Ensifer sp. NFY8; T3: Kocuria sp. RTL99; T4:

Mesorhizobium sp. NTY7; T5: Serratia sp. 5D; T6: Mesorhizobium sp. NTY7 + Ensifer

sp. NFY8; T7: Mesorhizobium sp. NTY7+ Kocuria sp. RTL99 and T8: Mesorhizobium

sp. NTY7+ Serratia sp. 5D


represent statistically different values and the capital letters in this table represent

overall effect of multiple factors like bacterial inoculation and plant type.





0.1134

3.3.4 Plant Growth Promoting Effect of Bacterial Inocula on

Chickpea Grown in Field (Year 2014-15)

Field experiments were conducted at NIBGE (District Faisalabad) to evaluate the effect

of three bacterial strains (Ensifer sp. NFY8, Mesorhizobium sp. NTY7 and Serratia

sp. 5D) as single-strain inocula for chickpea (Figure 3.24) as well as co-inoculation of

Mesorhizobium sp. NTY7 with Ensifer sp. NFY8 and Serratia sp. 5D. The results

revealed that all inoculated bacterial strains significantly increased the number of

nodules, weight of dry nodule, grain and straw yield of the both chickpea varieties

compared to non-inoculated control (Table 3.17, 3.18; Figure 3.25). The comparison of

the overall effect of inoculation on tested varieties indicated that the maximum increase

in grain yield was 1537.5 kg/ha recorded in the treatment inoculated with consortia of

Mesorhizobium sp. NTY7 + Ensifer sp. NFY8 which was significantly higher than that

3. Results

61

of the non-inoculated control (i.e., 1285.4 kg/ha). Among the bacterial strains, consortia

of Mesorhizobium sp. NTY7 + Ensifer sp. NFY8 was the most effective inoculant,

causing up to 19.61 % and 18.24 % increase in grain and straw yield, respectively over

non-inoculated control (Table 17, 18; Figure 3.25). Among the chickpea varieties

tested, Desi-type gave significantly higher yield in general (1681.5 and 1887.9 kg/ha

grain and straw, respectively) than Kabuli-type (1264.0 and 1877.4 kg/ha grain and

straw, respectively), which was true for inoculated as well as non-inoculated treatments.

Irrespective of chickpea variety and the bacterial inoculants, the number of nodules, dry

weight of nodule, grain yield and straw yield were found to be higher in all treatments

as compared to non-inoculated control (Table 3.17, 3.18; Figure 3.25).

3.3.5 Plant Growth Promoting Effect of Bacterial Inocula on

Chickpea Grown at Different Locations (Year 2015-16)

Field experiments were conducted at NIBGE and AARI experimental field (District

Faisalabad), AZRI Bhakkar and PRSS Kalurkot located in the Thal desert (District

Bhakkar) to evaluate the effect of two bacterial strains (Ensifer sp. NFY8 and

Mesorhizobium sp. NTY7) as single-strain inocula as well as co-inoculants for chickpea

(Figure 3.26). Soil analysis indicated that the soil at the Thal desert (AZRI Bhakkar and

PRSS Kalurkot) was nutrient-deficient compared with the NIBGE and AARI

experimental field (Table 3.19). Two chickpea varieties, i.e., Punjab 2008 (Desi-type)

and Noor 2009 (Kabuli-type) were used as host plants for inoculation studies at both

the experimental sites. The results revealed that inoculation with the bacterial strains

significantly increased the number of nodules, weight of dry nodules, grain yield and

straw yield of the tested crop varieties compared to non-inoculated control at all

localities (Table 3.20, 3.22, 3.23; Figure 3.27, 3.28). The comparison of the overall

effect of inoculation on tested varieties indicated that the maximum increase in grain

yield was 1232.2 kg/ha recorded in the treatment in which Mesorhizobium sp. NTY7

and Ensifer sp. NFY8 were used as co-inoculants. This yield was significantly higher

than that of the non-inoculated control (i.e., 1024.3 kg/ha). Among the bacterial strains,

co-inoculation of Mesorhizobium sp. NTY7 and Ensifer sp. NFY8 was the most

effective inoculant, causing up to 20.29 % and 14.60 % increase in grain and straw

yield, respectively over non-inoculated control (Table 3.20, 3.22, 3.23; Figure 3.27,

3.28). Chickpea variety Desi-type gave significantly higher yield in general (1274.8 and

1820.9 kg/ha grain and straw, respectively) than Kabuli-type (1060.6 and 1754.0 kg/ha

3. Results

62

grain and straw, respectively), which was true for inoculated as well as non-inoculated

treatments.

Among the sites used for cultivation of chickpea, higher yields of grain were

obtained at NIBGE (1443 kg/ha), followed by AZRI (1355.7 kg/ha), AARI (1292.9

kg/ha) and PRSS (579.1 kg/ha). Similarly, more straw yield was obtained at AZRI

(2603.5 kg/ha), followed by NIBGE (1846.7 kg/ha), AARI (1628.3 kg/ha) and PRSS

(1071.3 kg/ha). Higher number of nodule were obtained at NIBGE (9.29 no./plant),

followed by AARI (9.27 no. /plant), AZRI (7.33 no. /plant) and PRSS (7.29 no. /plant).

Similarly, more dry weight of nodules was obtained at AARI (230.00 mg/plant),

followed by NIBGE (213.3 mg/plant), AZRI (202.08 mg/plant) and PRSS (146.04

mg/plant). Irrespective of chickpea variety, experimental area and the bacterial

inoculants, the number of nodules, weight of dry nodule, grain yield and straw yield

were found to be higher in all treatments as compared to non-inoculated control (Table

3.20, 3.22, 3.23; Figure 3.27, 3.28).


field at NIBGE, Faisalabad. (Year 2014-15)

3. Results

63

Figure 3-25 Effect of bacterial inoculation on chickpea grown in field.

A= Numbers of nodules per plant, B= Weight of dry nodules per plant, C= Grain

yield per hectare and D= Straw yield per hectare. Strains used in this study: Ensifer

sp. NFY8, Mesorhizobium sp. NTY7, Serratia sp. 5D, Mesorhizobium sp. NTY7 +

Ensifer sp. NFY8 and Mesorhizobium sp. NTY7 + Serratia sp. 5D. Values are

average of 3 replicates. Error bars represent the standard deviations (SD). (Year 2014-

15)


field at different localities. (Year 2015-16)

3. Results

64


A= Numbers of nodule per Desi-type chickpea plant, B= Numbers of nodule per

Kabuli-type chickpea plant, C= Weight of dry nodules per Desi-type chickpea plant

and D= Weight of dry nodules per Kabuli-type chickpea plant. Strains used in this

study: Ensifer sp. NFY8, Mesorhizobium sp. NTY7 and Mesorhizobium sp. NTY7 +

Ensifer sp. NFY8. Values are average of 3 replicates. Error bars represent the standard

deviations (SD). (Year 2015-16)


3. Results

65

A= Grain yield (kg/ha) of Desi-type chickpea, B= Grain yield (kg/ha) of Kabuli-type

chickpea, C= Straw yield (kg/ha) of Desi-type chickpea and D= Straw yield (kg/ha)

of Kabuli-type chickpea. Strains used in this study: Ensifer sp. NFY8, Mesorhizobium

sp. NTY7 and Mesorhizobium sp. NTY7 + Ensifer sp. NFY8. Values are average of 3

replicates. Error bars represent the standard deviations (SD). (Year 2015-16)

Table 3.17 Effect of bacterial inoculation on number of nodules and dry

weight of nodules (mg/plant) of chickpea grown at experimental field. (Year

2014-15)


treatments

No. of

nodules

per plant

Dry weight

of nodules

per plant

No. of

nodules

per plant

Dry weight of

nodules per

plant

No. of

nodules

per plant

Dry weight

of nodules

per plant

T1 8.00 c 176.00 e 7.67 c 214.67 bcde 7.83 C 195.33 C

T2 9.00 abc 198.00 de 9.00 abc 252.00 abc 9.00 ABC 225.00 ABC

T3 9.33 abc 205.33 cde 9.33 abc 261.33 ab 9.33 AB 233.33 AB

T4 8.33 bc 183.33 e 8.67 abc 242.67 abcd 8.50 BC 213.00 BC

T5 10.00 ab 220.00 bcde 10.33 a 289.33 a 10.17 A 254.67 A

T6 9.00 abc 198.00 de 9.33 abc 261.33 ab 9.17 ABC 229.67 ABC

8.94 A 196.78 B 9.05 A 253.56 A

T: Treatments; T1: Control; T2: Ensifer sp. NFY8; T3: Mesorhizobium sp. NTY7; T4:

Serratia sp. 5D; T5: Mesorhizobium sp. NTY7 + Ensifer sp. NFY8 and T6:

Mesorhizobium sp. NTY7+ Serratia sp. 5D.

Values are average of 3 replicates. Different small letters in the same column represent

statistically different values and the capital letters represent overall effect of multiple

factors like bacterial inoculation and plant type.





LSD @ 0.05α for dry weight of nodules X type of chickpea X Treatments = 52.077

3. Results

66

Table 3.18 Effect of bacterial inoculation on grain and straw yield (kg/ha) of

chickpea grown at experimental field. (Year 2014-15)

T Desi-Type Chickpea Kabuli-Type

Chickpea

Overall effect of

treatments

Grain

yield

Straw

yield

Grain

yield

Straw

yield

Grain

yield

Straw

yield

T1 1106.4 e 1681.7 d 1106.4 e 1650.0 d 1285.4 C 1665.8 C

T2 1286.3 d 1905.0 c 1286.3 d 1904.0 c 1500.7 B 1904.5 B

T3 1294.3 d 1923.3 bc 1294.3 d 1919.3 c 1505.3 B 1921.3 B

T4 1281.7 d 1904.7 c 1281.7 d 1898.0 c 1491.7 B 1901.3 B

T5 1311.7 d 1973.0 a 1311.7 d 1966.3 ab 1537.5 A 1969.7 A

T6 1303.3 d 1939.7 abc 1303.3 d 1926.7 abc 1515.7 AB 1933.2 B

1681.5 A 1887.9 A 1264.0 B 1877.4 A


Serratia sp. 5D; T5: Mesorhizobium sp. NTY7 + Ensifer sp. NFY8 and T6:

Mesorhizobium sp. NTY7+ Serratia sp. 5D.








46.398

3. Results

67

Table 3.19 Characteristics of field soil from different localities*

Parameter AZRI

(District

Bhakkar)

PRSS

(District

Bhakkar)

AARI

(District

Faisalabad)

NIBGE

(District

Faisalabad)

Latitudea 31°38'09.2"

N

32° 09' 27.8"

N

31° 23' 48.8"

N

31° 23' 42.9"

N

Longitudea 71°07'16.3"

E

71° 16' 43.7"

E

73° 03' 12.3"

E

73° 01' 36.6" E

Altitudeb (m) 169 192 182 183

Annual rainfall

(Mean)c (mm)

300 150 350 350

Rainfall during

crop seasonc

(mm)

100 30 100 100

Date of Sowing October October November November

Date of

Harvesting

April April April April

Annual

Temperaturc

(°C)

0-50 0-50 0-50 0-50

Temperature

during crop

seasonc (°C)

0-40 0-40 0-40 0-40

Sandd (%) 73±1.5 70±1.5 44±0.5 41±1.5

Siltd (%) 18±2 20±1.5 28±1.5 30±1

Clayd (%) 9±1.5 10±1 28±2 29±1

Soil textured Sandy Loam Sandy Loam Clay loam Clay loam

Organic matterd

(%)

0.29±0.034 0.294±0.03 0.7±0.055 0.6±0.065

pH d 8.06±0.09 8.06±0.094 8.1±0.07 8.1±0.057

Electrical

conductivityd

(dS/m)

0.396±0.032 0.396±0.022 0.41±0.015 0.402±0.025

Total Pd (µg/g) 559±5.5 600±5.1 1106±20.0 1156±20.7

Available Pd

(µg/g)

3.139±0.25 3.20±0.30 7.82±0.6 7.92±0.6

Available Kd

(µg/g)

59.6±4.26 60.6±4.26 191.2±19.77 191.2±19.8

Available Nd

(%)

0.004±0.003

0

0.0042±0.003

8

0.0087±0.045 0.0087±0.040

* Samples collected at time of sowing. Source a= Google Earth, b= Soil survey of

Pakistan, c= Pakistan metrological department and d= this study

3. Results

68

Table 3.20 Effect of bacterial inoculation on nodulation of chickpea grown in

experimental fields at different locations. (Year 2015-16)

T Desi-Type Chickpea Kabuli-Type Chickpea Overall

effect L1 L2 L3 L4 L1 L2 L3 L4

T1 8.00

efg

8.67

cde

5.33 i 6.00

hi

8.33

def

7.33

efgh

5.33 i 5.00 i 6.75 C

T2 8.33

def

9.00

bcde

7.33

efgh

6.33

ghi

8.33

def

8.00

efg

6.67

fghi

7.33

efgh

7.67 B

T3 10.00

abcd

10.00

abcd

8.00

efg

7.67

efgh

10.00

abcd

10.00

abcd

8.33

def

8.67

cde

9.08 A

T4 11.00

a

10.33

abc

8.67

cde

8.67

cde

10.33

abc

10.67

ab

8.67

cde

9.00

bcde

9.67 A

9.33 A 9.50 A 7.33 B 7.17 B 9.25 A 9.00 A 7.25 B 7.50 B

8.33 A 8.25 A


Mesorhizobium sp. NTY7 + Ensifer sp. NFY8; L1: NIBGE; L2: AARI; L3: PRSS and

L4: AZRI



multiple factors like bacterial inoculation, location and plant type. LSD @ 0.05α for no

of nodules X type of chickpea= 0.4840, LSD @ 0.05α for no of nodules X Treatments

= 0.6845, LSD @ 0.05α for no of nodules X type of chickpea X locations = 0.9681 and

LSD @ 0.05α for no of nodules X type of chickpea X locations X Treatments = 1.9362.

Table 3.21 Effect of bacterial inoculation on dry weight of nodules per plant

(mg/plant) of chickpea grown in experimental fields at different locations. (Year

2015-16)



T1 168.67

ijklm

190.67

fghijkl

115.00

no

150.00

lmno

205.00

fghij

205.33

fghij

106.67

o

160.00

klm

162.67

C

T2 175.00

ghijklm

198.00

fghijk

146.67

mno

158.33

klmn

208.33

efghi

221.67

def

136.67

mno

213.33

efgh

182.25

B

T3 210.00

efghi

218.33

defg

160.00

klm

191.67

fghijkl

250.00

bcde

280.00

ab

163.33

jklm

260.00

abcd

216.67

A

T4 231.67

cdef

227.33

cdef

170.00

hijklm

216.67

defg

258.33

abcd

298.67

a

170.00

hijklm

266.67

abc

229.92

A

196.33

DE

208.58

CD

147.92

F

179.17

E

230.42

AB

251.42

A

144.17

F

225.00

BC

183.00 B 212.75 A

3. Results

69



L4: AZRI



multiple factors like bacterial inoculation, location and plant type.

LSD @ 0.05α for dry weight of nodules X type of chickpea= 10.892, LSD @ 0.05α for

dry weight of nodules X Treatments = 15.403, LSD @ 0.05α for dry weight of nodules

X type of chickpea X locations = 21.783 and LSD @ 0.05α for dry weight of nodules

X type of chickpea X locations X Treatments = 43.566.

Table 3.22 Effect of bacterial inoculation on grain yield (kg/ha) of chickpea

grown in experimental fields at different locations. (Year 2015-16)



T1 1431.0

d

1405.0

d

513.0

l

1083.0

g

1105.0

g

828.7 i 505.7

l

1323.0

ef

1024.3

C

T2 1681.7

b

1688.0

b

594.7

jk

1300.3

ef

1285.0

f

1003.0

h

588.3

k

1488.3

c

1203.7

B

T3 1720.0

ab

1688.3

b

595.0

jk

1308.3

ef

1290.0

ef

1006.7

h

588.3

k

1488.0

c

1210.6

B

T4 1728.0

a

1701.0

ab

631.3

j

1328.3

e

1303.3

ef

1022.7

h

616.7

jk

1526.3

c

1232.2

A

1640.2

A

1620.6

B

583.5

F

1255.0

D

1245.8

D

965.3

E

574.7

F

1456.4

C

1274.8 A 1060.6 B



L4: AZRI





Treatments = 13.574, LSD @ 0.05α for yield X type of chickpea X locations = 19.196

and LSD @ 0.05α for yield X type of chickpea X locations X Treatments = 38.392.

3. Results

70

Table 3.23 Effect of bacterial inoculation on straw yield (kg/ha) of chickpea

grown in experimental fields at different locations. (Year 2015-16)



T1 1634.7

k

1418.3

l

991.3

p

2553.3

b

1646.7

jk

1416.7

l

2998.0

p

2343.3

e

1625.3

D

T2 1880.0

h

1691.7

i

1065.7

o

2791.7

a

1889.7

gh

1688.3

ij

1073.0

o

2471.7

d

1819.0

C

T3 1924.7

fg

1699.7

i

1078.3

no

2808.0

a

1921.7

fgh

1698.0

i

1118.7

mn

2493.3

cd

1842.8

B

T4 1939.7

f

1707.7

i

1118.3

mn

2831.7

a

1936.3

f

1706.0

i

1127.0

m

2535.0

bc

1862.7

A

1844.7

C

1629.3

D

1063.4

E

2746.2

A

1848.6

C

1627.3

D

1079.2

E

2460.8

B

1820.9 A 1754.0 B



L4: AZRI




LSD @ 0.05α for straw X type of chickpea= 10.996, LSD @ 0.05α for straw X

Treatments = 15.550, LSD @ 0.05α for straw X type of chickpea X locations = 21.992

and LSD @ 0.05α for straw X type of chickpea X locations X Treatments = 43.983.

3. Results

71

3.4 Bacterial Diversity by Culture-Independent Molecular

Approach

3.4.1 Extraction of DNA and PCR Amplification of 16S rRNA and

nifH Genes from the Root Nodules and Rhizospheric Soil of

Chickpea

DNA was successfully extracted from the root nodules and rhizospheric soil of Kabuli-

type and Desi-type chickpea using bead beater machine. DNA of PCR-amplified 16S

rRNA and nifH genes was visualized on 1 % agarose gel (Figure 3.29).

Figure 3-29 Agarose gels showing DNA extracted from root nodules and PCR

amplification of 16S rRNA and nifH genes

A=DNA extraction from root nodules. Lane 1: Desi-type chickpea nodules; Lane 2:

Kabuli-type chickpea; B=PCR-amplification of 16S rRNA gene from nodule DNA of

chickpea. Lane 1: 1 kb ladder (Fermentas, Germany); Lane 2: Kabuli-type chickpea

(NIBGE); Lane 3: Desi-type chickpea (NIBGE); Lane 4: Desi-type chickpea (Thal

desert) and Lane 5: Negative control and C= PCR-amplification of partial nifH gene

from nodule DNA of chickpea. Lane 1: Kabuli-type chickpea (NIBGE); Lane 2: Desi-

type chickpea (NIBGE); Lane 3: Desi-type chickpea (Thal desert); Lane 4: Negative

control and Lane 5: 1 kb ladder (Fermentas, Germany)


Analysis of nifH gene Amplified from Nodule DNA

Sequence analysis of nifH gene PCR-amplified from nodule DNA revealed diversity of

diazotrophic (nitrogen-fixing) bacteria associated with the nodules of chickpea. Overall

62,670 sequences of nifH gene were retrieved from the nodules of chickpea collected

from different sites which included 13,720 sequences from Kabuli-type and 11,482

3. Results

72

sequences from Desi-type NIBGE. Among the obtained sequences, 20,020 sequences

were retrieved from Thal desert, 7,427 sequences from Chowk Munda, 5,976 sequences

from Kallar Syedan and 4,045 sequences from NIFA. A significant fraction i.e., 88.83

% of the total sequences retrieved from all sites belonged to genus Mesorhizobium. nifH

gene sequences belonging to diazotrophic genera Bradyrhizobium, Frankia and

Paenibacillus were also detected. nifH gene sequences of “uncultured” bacteria

comprised 0.12 % of the total recovered sequences. Other minor OTU represented by

≤ 26 sequences formed 10.99 % of total recovered and remained unidentified (Table

3.24). In the present study, four type of mesorhizobial sequences were detected among

the retrieved sequences of mesorhizobia (Table 3.25). The sequences of Mesorhizobium

mediterraneum were most abundant (56.08 %) as compared to sequences of

Mesorhizobium ciceri (39.83 %), Mesorhizobium septentrionale (3.98 %) and

Mesorhizobium huakuii (0.09 %). nifH sequences of Mesorhizobium septentrionale and

Mesorhizobium huakuii were detected only in the nodules collected from NIFA and

Thal desert.

Table 3.24 Dominant bacterial genera detected by nifH gene sequences

amplified from nodules of chickpea grown at different localities

Genera

detected

L1 L2 L3 L4 L5 L6 L7

Bradyrhizobium 3 (0.02

%)

1 (0.01

%)

0 0 0 0 4 (0.01

%)

Frankia 0 0 9 (0.04

%)

0 0 0 9 (0.01

%)

Mesorhizobium 12076

(88.02

%)

10693

(93.14

%)

17033

(85.08

%)

5362

(89.73

%)

3527

(87.19

%)

6977

(93.95

%)

55668

(88.83

%)

Paenibacillus 0 0 29 (0.14

%)

0 0 0 29 (0.05

%)

Uncultured

nitrogen-fixing

bacteria

7 (0.05

%)

4 (0.03

%)

9 (0.04

%)

53 (0.89

%)

0 0 73 (0.12

%)

Other minor

O.T.U. ≤ 26

1634

(11.91

%)

783

(6.82 %)

2940

(14.69

%)

561

(9.39 %)

518

(12.81

%)

449

(6.05 %)

6885

(10.99

%)

L1: NIBGE Kabuli-type; L2: NIBGE Desi-type; L3: Thal desert Desi-type; L4: Kallar

Syedan Desi-type; L5: NIFA Desi-type; L6: Chowk Munda Desi-type and L7: Overall

Nodules

3. Results

73

Table 3.25 Mesorhizobial sequences detected by nifH gene amplification from

nodules of chickpea grown at different localities

Species

detected

L1 L2 L3 L4 L5 L6 L7

M. ciceri 2654

(21.98

%)

2630

(24.60

%)

8555

(50.23 %)

2437

(45.45

%)

1364

(38.67

%)

4538

(65.04

%)

22178

(39.84

%)

M. huakuii 0 0 46 (0.27

%)

0 6 (0.17

%)

0 52 (0.09

%)

M.

mediterraneum

9422

(78.02

%)

8063

(75.40

%)

6363

(37.36%)

2925

(54.55

%)

2008

(56.93

%)

2439

(34.96

%)

31220

(56.08

%)

M.

septentrionale

0 0 2069

(12.15 %)

0 149

(4.22 %)

0 2218

(3.98 %)



Nodules

3.4.3 Bacterial Diversity Revealed by Sequence Analysis of nifH Gene

Amplified from Rhizospheric Soil DNA

Sequence analysis of nifH gene PCR-amplified from rhizospheric soil DNA revealed

diversity of diazotrophic (nitrogen-fixing) bacteria associated with chickpea. A total of

47,157 sequences of nifH gene were retrieved from the rhizospheric and non-

rhizospheric soil of chickpea collected from different sites which included 5,875

sequences from Kabuli-type and 3,889 sequences from Desi-type grown at NIBGE. The

sequences retrieved from rhizospheric soil of Desi-type chickpea included, 3,121

sequences from Thal desert, 6,135 sequences from Chowk Munda, 6,826 sequences

from Kallar Syedan and 8,922 sequences from NIFA. Sequences of nifH gene were also

retrieved from the bulk soil of chickpea collected from two sites which included 5,450

sequences from NIBGE and 6,939 sequences from NIFA. A significant fraction i.e.,

16.68 % of the total nifH sequences retrieved from all sites belonged to genus

Mesorhizobium. In the present study 34.63 % sequences belonging to 5 genera of

culturable diazotrophic bacteria (Mesorhizobium, Bradyrhizobium, Ensifer, Frankia

and Azatobactor) were retrieved along with 20.68 % sequences of “uncultured”

bacteria. Other minor OTU represented by ≤ 26 sequences formed 57.18 % of total

which remained unidentified (Table 3.26).

In the present study four type of mesorhizobial sequences were detected among

the retrieved sequences of mesorhizobia (Table 3.27). The sequences of Mesorhizobium

3. Results

74

mediterraneum were most abundant (57.53 %) as compared to sequences of

Mesorhizobium ciceri (31.45 %), Mesorhizobium septentrionale (10.68 %) and

Mesorhizobium huakuii (0.34 %). nifH sequences of Mesorhizobium huakuii were

detected only in the nodules collected from NIFA.

Table 3.26 Bacterial genera detected by nifH gene amplification from

rhizospheric soil of chickpea grown at different localities

Genera

detected

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

Anaeromyxobac

ter

0 29

(0.42

%)

0 0 0 0 0 0 29

(0.23

%)

0

Azospirillum 16

(0.29

%)

6

(0.09

%)

36

(1.11

%)

25

(0.39

%)

0 1

(0.01

%)

0 18

(0.31

%)

22

(0.18

%)

80

(0.21

%)

Azotobacter 11

(0.20

%)

3

(0.04

%)

62

(1.91

%)

482

(7.58

%)

0 16

(0.15

%)

7

(0.18

%)

7

(0.12

%)

14

(0.11

%)

574

(1.54

%)

Bradyrhizobium 61

(1.09

%)

7

(0.10

%)

26

(0.80

%)

153

(2.41

%)

24

(0.35

%)

12

(0.11

%)

56

(1.43

%)

45

(0.77

%)

68

(0.54

%)

316

(0.85

%)

Chlorogloeopsis 0 39

(0.56

%)

0 0 0 0 0 0 39

(0.31

%)

0

Frankia 28

(0.50

%)

0 392

(12.0

7%)

480

(7.55

%)

0 0 0 0 28

(0.22

%)

872

(2.34

%)

Mesorhizobium 312

(5.59

%)

6

(0.09

%)

386

(11.8

8 %)

567

(8.92

%)

553

(7.95

%)

4586

(42.0

5 %)

103

(2.62

%)

20

(0.34

%)

318

(2.54

%)

6215

(16.68

%)

Ensifer 10

(0.18

%)

22

(0.32

%)

0 137

(2.16

%)

8

(0.12

%)

6

(0.06

%)

28

(0.71

%)

17

(0.29

%)

32

(0.26

%)

196

(0.53

%)

Uncultured

nitrogen-fixing

bacteria

2280

(40.8

5 %)

1728

(24.9

1 %)

897

(27.6

2 %)

2497

(39.2

8 %)

1461

(21.0

0 %)

1501

(13.7

6 %)

443

(11.2

9 %)

907

(15.4

3 %)

4008

(32.0

2 %)

7706

(20.68

%)

Other minor

O.T.U. ≤ 26

2864

(51.3

1 %)

5097

(73.4

9 %)

1449

(44.6

1 %)

2016

(31.7

1 %)

4910

(70.5

9 %)

4785

(43.8

7 %)

3287

(83.7

7 %)

4864

(82.7

5%)

7961

(63.5

9%)

21311

(57.18

%)

L1: Bulk Soil NIBGE; L2: Bulk Soil NIFA; L3: Thal desert Desi-type; L4: Chowk

Munda Desi-type; L5: Kallar Syedan Desi-type; L6: NIFA Desi-type; L7: NIBGE

Desi-type; L8: NIBGE Kabuli-type; L9: Overall Bulk Soil and L10: Overall

rhizospheric soil

3. Results

75

Table 3.27 Mesorhizobial sequences detected by nifH gene amplified from

rhizospheric soil of chickpea grown at different localities

Species

detected L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

M. ciceri 48

(26.6

7 %)

4

(80.0

0 %)

132

(50.9

7 %)

123

(35.6

5 %)

279

(67.0

7 %)

606

(23.3

4 %)

21

(33.8

7 %)

6

(46.1

5 %)

52

(28.1

1 %)

1219

(31.4

5 %)

M. huakuii 0 0 0 0 0 13

(0.50

%)

0 0 0 13

(0.34

%)

M.

mediterrane

um

128

(71.1

1 %)

0 72

(27.8

0 %)

217

(62.9

0 %)

137

(32.9

3 %)

1628

(62.7

1 %)

41

(66.1

3 %)

7

(53.8

5 %)

128

(69.1

9 %)

2230

(57.5

3 %)

M.

septentriona

le

4

(2.22

%)

1

(20.0

0 %)

55

(21.2

4 %)

5

(1.45

%)

0 349

(13.4

4 %)

0 0 5

(2.70

%)

414

(10.6

8 %)




rhizospheric soil.


Analysis of 16S rRNA Gene Amplified from Nodule DNA

Pyrosequencing of the 16S rRNA gene PCR amplified from nodule DNA revealed

bacterial diversity in the root nodules of chickpea. Overall 37,229 sequences were

retrieved from the nodules of chickpea collected from different sites which included

11,398 sequences from Kabuli-type (NIBGE), 8,690 sequences from Desi-type

(NIBGE), 9,672 sequences from Thal desert, 2,737 sequences from Chowk Munda,

2,743 sequences from Kallar Syedan and 1,989 sequences from NIFA. The retrieved

sequences belonged to diverse phyla (Table 3.28) including Proteobacteria (90.41 %),

Firmicutes (4.93 %), Actinobacteria (0.76 %) and “uncultured” phyla (3.23 %).

Further analysis of the retrieved sequences indicated that retrieved sequences

belonged to diverse taxa (Table 3.29; Figure 3.30) including α-Proteobacteria (56.99

%), γ-Proteobacteria (26.43 %), “uncultured” (7.67 %), Firmibacteria (4.93 %), β-

Proteobacteria (2.55 %), Actinobacteria (0.75 %) and other minor groups (0.66 %).

Comparison of the retrieved sequences from different localities revealed that at the sites

Kallar Syedan and Chowk Munda, α-proteobacterial sequences were relatively under

represented (i.e., 15.29 % and 25.91 % of the total sequences, respectively) compared

to NIBGE experimental site (75.63 %) but at both sites maximum number of γ-

3. Results

76

Proteobacterial sequences (54.77 % and 52.14 %, respectively) were detected. Class

Firmibacteria was not represented in the retrieved sequences from the site Kallar

Syedan and NIFA.

Further analysis of the retrieved sequences indicated that sequences of mostly

the bacterial genera isolated in the present study were retrieved along with numerous

sequences belonging to diverse genera of culturable as well as “uncultured” bacteria

(Appendix A; Figure 3.31). A significant fraction i.e., 52.77 % of the total sequences

retrieved from all sites belonged to genus Mesorhizobium (Figure 3.31). In the nodules

of Desi-type chickpea cultivated in experimental field at NIBGE sequences of the genus

Mesorhizobium were more abundant (71.98 % of the total sequences) as compared to

Kallar Syedan and Chowk Munda where the mesorhizobial sequences comprised about

12.49 % and 22.35 % of the total sequences, respectively. In the present study 70.78 %

sequences belonging to 111 genera of culturable bacteria were retrieved along with

29.22 % sequences of “uncultured” bacteria.

Detection of Serratia spp. in the Root Nodules by Culture-Independent DNA-Based

Technique (16S rRNA Sequence Analysis)

Detailed characterization of root nodule endophytic microbial communities of chickpea

suggested high dominance of Serratia related sequences in the root nodules of Desi and

Kabuli-types grown in the NIBGE (District Faisalabad) and the Thal desert. In the Desi-

type cultivar grown in Thal desert up to 15 % of total bacterial sequences detected in

the root nodules were related to genus Serratia. Whereas, both Desi and Kabuli-types

grown in the district Faisalabad showed that about 2 % of total bacterial sequences from

the root nodules of each type belonged to the genus Serratia (Figure 3.32). Overall

Serratia affiliated sequences in root nodules collected from both sites could be grouped

into two major clusters at 98 % DNA similarity. The most dominant group or cluster

represented by 1,093 Serratia sequences showed high similarity with the Serratia

marcescens sp. whereas other cluster represented by only 43 sequences was unique and

did not show close similarity with any cultured Serratia strains at 98 % DNA similarity.

The separation between these two Serratia clusters was supported through high

bootstraps values and suggested presence of a potential novel Serratia sp. that has not

been described previously. Overall clustering of other Serratia species was similar for

the short 375 bp fragment of 16S rRNA gene as compared to complete 16S rRNA gene

fragment (Figure 3.32).

3. Results

77

Comparison of the relative abundance of Serratia affiliated sequences in

relation to geochemical characteristics at the two sites (NIBGE and Thal desert)

suggested a high abundance of Serratia sequences was strongly associated with the

nodules from Thal desert (District Khushab) and that site was significantly lower in

both total and available P. Whereas N contents were similar at both sites and more

variation in N contents were noticed within sites as compared to among both sites

(Figure 3.33).

Figure 3-30 Relative abundance of major bacterial classes detected by 16S

rRNA gene sequence analysis in the root nodules of chickpea grown at different

localities

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

α-Proteobacteria γ-Proteobacteria Unclassified β-Proteobacteria

Actinobacteria Firmibacteria Others

3. Results

78

Identification of the Isolated Serratia Strains from Root Nodules of Chickpea

Reddish pigment producing bacterial strains 5D and RTL100 were isolated from the

nodules of chickpea collected from the NIBGE (District Faisalabad) fields and rainfed

Thal desert (District Khushab), respectively. On LB agar plates both the isolates formed

brick red colonies with entire margins and the cells were motile, rod shaped and Gram

negative. Both isolates have been identified as members of the genus Serratia based on

the 16S rRNA gene sequencing. Bacterial isolates 5D and RTL 100 showed 100 % and

99.6 % sequence homology with the 16S rRNA gene of Serratia marcescens strains

DSM 30121T and JCM 1239, respectively (Figure 3.34).

Figure 3-31 Relative abundance of the dominant bacterial genera detected by

16S rRNA gene sequence analysis in nodules of chickpea grown in different

localities

i.e., A=Overall, B=Kabuli-type NIBGE, C=Desi-type NIBGE, D=Thal desert,

E=Chowk Munda, F=NIFA and G=Kallar Syedan.

http://www.dsmz.de/catalogues/details/culture/DSM-30121.html

3. Results

79

Figure 3-32 Molecular phylogenetic analysis of the 16S rRNA sequences

retrieved from root nodules of chickpea.

The tree was constructed by Maximum Likelihood method. Only maximum

likelihood bootstrap node support values ≥50 are shown at the nodes. Total number of

Serratia-related sequences amplified from the root nodules of different plant varieties

and their relative proportion to the total number of sequences amplified from root

nodules have been presented in the Table.

Figure 3-33 Non-metric multi-dimensional scaling representation of the

geochemical characteristics and relative abundance of the Serratia sequences in

the root nodules of chickpea of NIBGE (District Faisalabad) and Thal desert

areas. Among all variables tested, these were significantly (P <0.05) associated with

two sites.

3. Results

80

Figure 3-34 16S rRNA sequence-based phylogenetic tree of Serratia strains

isolated from root nodule of chickpea constructed by maximum likelihood

method. Only maximum likelihood bootstrap node support values ≥50 are shown at

the nodes.


sequence analysis in the root nodules of chickpea grown at different localities.

(Data shown in percentage)

Phyla detected Kabuli-

NIBGE

Desi-

NIBGE

Thal

desert

Kallar

Syedan

NIFA Chowk

Munda

Overall

Acidobacteria 0.02 0.00 0.00 0.00 0.00 0.00 0.006

Actinobacteria 1.78 0.45 0.29 0.17 0.22 0.33 0.76

Bacteroidetes 1.09 0.44 0.01 0.97 0.29 1.81 0.62

Chloroflexi 0.01 0.01 0.00 0.00 0.00 0.00 0.006

Firmicutes 0.24 0.04 15.92 0.00 0.00 0.71 4.93

Gemmatimonadetes 0.00 0.01 0.00 0.00 0.00 0.00 0.003

Planctomycetes 0.05 0.00 0.01 0.00 0.00 0.00 0.02

Proteobacteria 94.32 96.03 80.52 97.02 96.49 87.10 90.41

Thaumarchaeota 0.03 0.01 0.00 0.00 0.00 0.00 0.01

Verrucomicrobia 0.00 0.03 0.00 0.00 0.00 0.00 0.006

Uncultured 2.46 2.96 3.25 1.85 3.00 10.04 3.23

3. Results

81

Table 3.29 Relative abundance of bacterial classes detected by 16S rRNA gene

sequence analysis from nodules of chickpea grown in different localities.


Classes detected L1 L2 L3 L4 L5 L6 L7

Acidobacteria_Gp3 0.01 0.00 0.00 0.00 0.00 0.00 0.003

Acidobacteria_Gp4 0.01 0.00 0.00 0.00 0.00 0.00 0.003

Actinobacteria 1.76 0.45 0.29 0.17 0.22 0.33 0.75

α-Proteobacteria 62.17 75.63 55.51 15.29 52.56 25.91 56.99

Anaerolineae 0.01 0.00 0.00 0.00 0.00 0.00 0.003

Bacilli 0.23 0.04 15.92 0.00 0.00 0.71 4.93

Bacteroidetes_incertae_sedi

s

0.06 0.01 0.00 0.00 0.00 0.00 0.02

β-Proteobacteria 0.77 0.34 1.66 17.81 5.93 2.14 2.55

Clostridia 0.01 0.00 0.00 0.00 0.00 0.00 0.003

Cytophagia 0.02 0.01 0.00 0.04 0.00 0.00 0.01

ƍ-proteobacteria 0.03 0.00 0.02 0.00 0.00 0.00 0.02

Flavobacteriia 0.15 0.25 0.00 0.50 0.00 0.11 0.14

γ-Proteobacteria 27.64 17.19 19.03 54.77 31.92 52.14 26.43

Gemmatimonadetes 0.00 0.01 0.00 0.00 0.00 0.00 0.003

Opitutae 0.00 0.03 0.00 0.00 0.00 0.00 0.01

Planctomycetia 0.05 0.00 0.01 0.00 0.00 0.00 0.02

Sphingobacteriia 0.82 0.16 0.01 0.42 0.29 1.65 0.42

Thermomicrobia 0.00 0.01 0.00 0.00 0.00 0.00 0.003

Uncultured 6.25 5.85 7.55 11.00 9.08 17.01 7.67



Nodules

3.4.5 Bacterial Diversity Revealed by Sequence Analysis of 16S rRNA

Gene Amplified from Rhizospheric Soil DNA

Overall 59,329 sequences of 16S rRNA gene were retrieved from the rhizospheric and

non-rhizospheric soil of chickpea collected from different sites. The sequences included

8962 sequences from rhizospheric soil of Kabuli-type and 10,223 sequences from Desi-

type grown at NIBGE. The sequences retrieved from Desi-type chickpea included

13,152 sequences from Thal desert, 5,550 sequences from Chowk Munda, 5,642

sequences from Kallar Syedan and 6,710 sequences from NIFA. 16S rRNA gene

sequences were retrieved from the non-rhizospheric soil of chickpea collected from two

sites which included 4,890 sequences from NIBGE and 4,200 sequences from NIFA.

The retrieved sequences belonged to diverse phyla including Proteobacteria (23.396

%), Firmicutes (5.516 %), Actinobacteria (29.479 %), “uncultured” (28.042 %) and

other minor phyla (13.567 %) (Table 3.30; Figure 3.35).

3. Results

82

Further analysis of the retrieved sequences from rhizospheric soil indicated that

retrieved sequences belonged to diverse taxa (Table 3.31) including α-Proteobacteria

(10.436 %), γ-Proteobacteria (3.489 %), “uncultured” (35.970 %), Firmibacteria

(4.99%), β-Proteobacteria (4.944 %), Actinobacteria (26.651 %) and other minor

groups (13.52 %).

Further analysis of the retrieved sequences from the rhizospheric soil indicated

that sequences of all the bacterial genera isolated in the present study were retrieved

along with numerous sequences belonging to diverse genera of culturable as well as

“uncultured” bacteria (Appendix B). Only a minor fraction i.e., 0.265 % of the total

sequences retrieved from all sites belonged to genus Mesorhizobium. In the

rhizospheric soil of Desi-type chickpea cultivated at Kallar Syedan sequences of the

genus Mesorhizobium were more abundant (0.815 % of the total sequences) as

compared to non-rhizospheric soil of chickpea at NIBGE where the mesorhizobial

sequences comprised about 0.041 % of the total sequences. In the present study 29.72

% sequences belonging to 313 genera of culturable bacteria were retrieved from

rhizospheric soil along with 70.28 % sequences of “uncultured” bacteria.

Figure 3-35 Relative abundance of major bacterial phyla detected by 16S

rRNA gene sequence analysis from rhizospheric soil of chickpea grown at

different localities.

3. Results

83


sequence analysis in the rhizospheric soil of chickpea grown at different

localities. (Data shown in percentage)

Phyla detected L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

Acidobacteria 0.798 5.929 2.502 1.063 4.833 3.785 4.040 4.407 3.168 3.410

Actinobacteria 26.52

4

17.85

7

38.40

5

41.91

0

28.85

0

25.97

6

20.76

7

22.28

3

22.51

9

29.479

Armatimonadetes 0.020 0.310 0.327 0.180 0.845 0.253 0.509 0.714 0.154 0.462

Bacteroidetes 6.094 1.952 1.118 3.658 3.032 1.952 2.827 3.135 4.180 2.420

BRC1 0.020 0.000 0.015 0.036 0.000 0.015 0.020 0.022 0.011 0.018

candidate division

WPS-1

0.000 0.000 0.038 0.036 0.147 0.045 0.108 0.056 0.000 0.068

candidate division

WPS-2

0.000 0.190 0.091 0.018 0.221 0.104 0.068 0.056 0.088 0.088

Candidatus

Saccharibacteria

0.000 0.000 0.038 0.000 0.018 0.045 0.020 0.011 0.000 0.024

Chloroflexi 0.654 2.238 0.814 0.288 1.286 1.013 2.436 2.723 1.386 1.501

Cyanobacteria/Chloro

plast

0.204 0.381 0.106 0.847 0.588 0.462 1.017 0.781 0.286 0.593

Deinococcus-Thermus 0.061 0.000 0.015 0.360 0.000 0.000 0.000 0.000 0.033 0.044

Euryarchaeota 0.061 0.024 0.144 0.108 0.000 0.015 0.010 0.056 0.044 0.064

Firmicutes 11.32

9

3.857 4.805 12.27

0

4.190 2.474 5.664 5.412 7.877 5.516

Gemmatimonadetes 1.329 0.976 0.403 0.198 0.496 0.715 0.489 0.580 1.166 0.480

Hydrogenedentes 0.000 0.000 0.000 0.000 0.000 0.000 0.010 0.000 0.000 0.002

Ignavibacteriae 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.011 0.000 0.002

Latescibacteria 0.000 0.000 0.000 0.000 0.000 0.015 0.000 0.000 0.000 0.002

Nitrospirae 0.184 0.714 0.335 0.288 0.294 0.387 0.470 0.469 0.429 0.382

Planctomycetes 2.025 2.929 2.623 0.793 1.378 2.265 3.316 3.247 2.442 2.480

Proteobacteria 16.15

5

19.31

0

19.92

1

22.30

6

28.79

5

29.40

4

23.98

5

21.24

5

17.61

3

23.396

Thaumarchaeota 0.654 4.048 0.852 0.378 1.011 1.818 1.682 1.852 2.222 1.290

Uncultured 33.74

2

38.95

2

27.40

3

15.18

9

27.10

4

29.34

4

32.23

1

32.71

6

36.15

0

28.042

Verrucomicrobia 0.143 0.333 0.046 0.072 0.588 0.358 0.333 0.223 0.231 0.239




rhizospheric soil

3. Results

84

Table 3.31 Relative abundance of major bacterial classes detected by 16S

rRNA gene sequence analysis from rhizospheric soil of chickpea grown at

different localities.


Classes detected L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 Acidobacteria_Gp1 0.020 0.048 0.046 0.000 0.165 0.015 0.137 0.179 0.033 0.092

Acidobacteria_Gp10 0.020 0.214 0.144 0.000 0.551 0.194 0.059 0.123 0.110 0.157











Actinobacteria 24.00

8

14.92

9

34.79

3

40.57

7

25.76

3

23.33

8

18.14

5

19.39

3

19.81

3

26.651

α-Proteobacteria 9.836 8.405 9.649 9.874 10.603

13.219

10.388

10.042

9.175 10.436

Anaerolineae 0.061 1.548 0.547 0.090 0.956 0.551 1.829 2.053 0.748 1.069

Armatimonadia 0.000 0.048 0.008 0.018 0.055 0.000 0.029 0.033 0.022 0.022

Bacilli 10.81

8

3.167 4.630 12.01

8

4.006 2.101 4.568 4.519 7.283 4.990

Bacteroidetes_incertae_se

dis

0.245 0.405 0.182 0.180 0.147 0.492 0.577 0.513 0.319 0.358

β-Proteobacteria 1.595 2.667 4.174 5.297 10.584

5.529 3.737 3.481 2.090 4.944

Caldilineae 0.102 0.286 0.030 0.018 0.147 0.119 0.235 0.156 0.187 0.117

Chloroflexia 0.000 0.000 0.008 0.000 0.074 0.045 0.088 0.179 0.000 0.066

Chloroplast 0.000 0.214 0.038 0.847 0.037 0.149 0.157 0.167 0.099 0.189

Chthonomonadetes 0.000 0.071 0.008 0.000 0.202 0.045 0.049 0.179 0.033 0.072

Clostridia 0.041 0.357 0.046 0.000 0.092 0.164 0.655 0.625 0.187 0.289

Cyanobacteria 0.204 0.167 0.068 0.000 0.496 0.253 0.704 0.524 0.187 0.342

Cytophagia 5.194 0.119 0.175 1.640 0.184 0.224 0.509 0.703 2.849 0.506

Dehalococcoidia 0.061 2.286 0.122 0.360 1.268 1.565 2.299 2.935 0.033 0.032

Deinococci 0.000 0.000 0.015 0.000 0.000 0.000 0.000 0.000 0.000 0.044

ƍ-proteobacteria 0.818 0.000 2.851 1.207 0.000 0.000 0.000 0.000 1.496 2.217

ɛ-proteobacteria 0.000 0.000 0.008 0.000 0.000 0.000 0.000 0.000 0.000 0.002

Erysipelotrichia 0.000 0.000 0.000 0.018 0.000 0.000 0.000 0.000 0.000 0.002

Flavobacteriia 0.000 0.214 0.053 0.000 0.055 0.089 0.088 0.201 0.099 0.086

γ-Proteobacteria 2.249 2.643 1.483 4.811 3.712 5.276 4.715 2.823 2.431 3.489

Gemmatimonadetes 1.329 0.976 0.403 0.198 0.496 0.715 0.489 0.580 1.166 0.480

Halobacteria 0.061 0.000 0.000 0.000 0.000 0.000 0.000 0.022 0.033 0.004

Ignavibacteria 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.011 0.000 0.002

Ktedonobacteria 0.000 0.000 0.000 0.000 0.018 0.030 0.000 0.000 0.000 0.006

Methanomicrobia 0.000 0.000 0.046 0.000 0.000 0.000 0.000 0.000 0.000 0.012

Negativicutes 0.000 0.119 0.000 0.000 0.000 0.000 0.166 0.011 0.055 0.036

Cont…

3. Results

85

Nitrospira 0.184 0.714 0.335 0.288 0.294 0.387 0.470 0.469 0.429 0.382

Opitutae 0.123 0.286 0.046 0.072 0.515 0.298 0.284 0.167 0.198 0.203

Planctomycetia 2.025 2.929 2.616 0.793 1.378 2.250 3.316 3.225 2.442 2.472

Sphingobacteriia 0.184 0.667 0.601 1.712 2.315 0.954 0.959 0.826 0.407 1.067

Subdivision3 0.020 0.048 0.000 0.000 0.055 0.060 0.039 0.056 0.033 0.032

Thermomicrobia 0.123 0.071 0.038 0.108 0.000 0.030 0.059 0.078 0.099 0.052

Thermoplasmata 0.000 0.000 0.008 0.054 0.000 0.000 0.010 0.022 0.000 0.014

Verrucomicrobiae 0.000 0.000 0.000 0.000 0.000 0.000 0.010 0.000 0.000 0.002

Uncultured 39.91

8

50.88

1

34.58

8

18.75

7

31.75

3

38.40

5

41.45

6

41.70

9

44.98

3

35.970




rhizospheric soil

86

4 Discussion

Diversity of culturable bacteria in the root nodules and rhizosperic soil of chickpea was

investigated by isolation on the growth media followed by identification on the basis of

16S rRNA sequence analysis. The study on bacterial diversity was extended to include

occurrence of “non-cultured” bacteria by PCR amplification of 16S rRNA and nifH

genes from nodules and rhizospheric soil DNA followed by sequence analysis. In the

present study 28 root nodule and rhizospheric soil samples and 6 samples of bulk soil

collected from 5 different sites were processed. In Pakistan farmers prefer to grow Desi-

type chickpea due to its disease resistance and high yield [113]. Therefore, nodules

from Desi-type were available from all the collection sites but nodules of Kabuli-type

chickpea were available only from the experimental field of NIBGE (District

Faisalabad).

Among the 60 bacterial isolates obtained in the present study, 23 endophytic

isolates were obtained from the nodules of chickpea. Among 23 endophytes, 10 strains

were identified by 16S rRNA gene sequence analysis as Mesorhizobium spp. and

further confirmed by their nodulating ability of original host chickpea. Only one type

of nifH and 16S rRNA sequence of Mesorhizobium was detected in pure cultures,

pointing to the possibility that purified mesorhizobial isolates are re-isolates of the same

strain. Presently members of only this genus are accepted as true nitrogen fixing

endosymbionts of chickpea nodules [3].

Fifty isolates of non-nodulating bacteria were obtained from the rhizosphere and

root nodules of chickpea. The isolates NTY29, NTY33, RTY42, JSN114, NTN143 and

NTN143 were obtained from the rhizosphere of chickpea growing in farmer field of

Thal desert and experimental field of NIBGE. All these strains were identified as

Bacillus spp. on the basis of 16S rRNA sequence analysis. There are several reports on

isolation of Bacillus from soils of this region [157] and also form desert of Kutch [158].

The isolates NFY126, NFY130 and RTN142 were purified from the rhizosphere of

chickpea grown in Kallar Syedan and Thal desert. The strains were identified as Bosea

87

sp. on the basis of 16S rRNA sequence analysis. Jaramillo et al. [159] have isolated and

characterised Bosea sp. from the nodules of cowpea (Vigna unguiculata). The isolate

NFY8 was obtained from the nodule of chickpea growing in field of NIBGE. This strain

was identified as Ensifer sp. on the basis of 16S rRNA sequence similarity. Appunu et

al. [160] have isolated Ensifer sp. from soybean (Glycine max L.). The isolates NTY34,

NTY38, NTY48 and NFY132 were obtained from the rhizosphere of chickpea growing

in field of NIBGE and Thal desert. These strains were identified as Enterobacter sp. on

the basis of 16S rRNA sequence similarity. Tahir et al. [105] have also isolated

Enterobacter sp. from the rhizosphere of wheat from this region. The isolates NTY36

and NFY121 were obtained from the rhizosphere of chickpea growing in field of

NIBGE. These strains were identified as Klebsiella sp. on the basis of 16S rRNA

sequence similarity. Ladha et al. [161] isolated nitrogen fixing Klebsiella sp. from the

rhizosphere of rice. The isolates RTL54 and RTL99 were obtained from the rhizosphere

of chickpea growing in farmer field of Thal desert and Chowk Munda. These strains

were identified as Kocuria sp. on the basis of 16S rRNA sequence similarity. Goswami

et al. [158] have isolated Kocuria sp. from saline desert of Kutch, India. The isolates

RSY14, NTY31, NTY39, RTY50, NTY51, NFY122, NTY123, NFY125, NFY134,

NTY139, NFN147 and NTN153 were obtained from the rhizosphere and nodules of

chickpea growing in farmer field of Thal desert, Chowk Munda, Kallar Syedan and

experimental field of NIFA and NIBGE. These strains were identified as Pseudomonas

sp. on the basis of 16S rRNA sequence similarity. Isolation of Pseudomonas sp. was

previously reported from nodules of different legumes [10]. as well as rhizosphere of

different crops including chickpea [162, 163].

Rhizobial isolates from nodules included 3 strains of Rhizobium and 2 strains

of Ensifer. Members of these two genera are known to be the nitrogen-fixing nodule

endophytes of different legumes [9]. The remaining 8 nodule endophytic isolates were

identified as members of genera Ochrobactrum, Paenibacillus, Pseudomonas and

Serratia. Isolation of all these bacterial genera have been previously reported from

nodules of different legumes [10, 13, 14, 114, 115]. However, exact role of these

bacteria still remains unclear. Previously Deng et al. [12] have also found the co-

occurrence of Paenibacillus and Pseudomonas with Mesorhizobium in the nodules of

Sphaerophysa salsula.

4. Discussions

88

Phylogenetic analysis of proteobacterial isolates indicated two diverse clusters

of Pseudomonas isolates. Seven Pseudomonas isolates formed cluster with

Pseudomonas hibiscicola ATCC 19867T (AB021405) and the remaining five

Pseudomonas isolates formed cluster with Pseudomonas taiwanensis BCRC 17751T

(EU103629). The isolates which deviate from the established Pseudomonas strains in

the phylogenetic tree may be transferred to a different genus upon further verification

as has been suggested previously [116]. In the present study, 60 bacterial isolates

obtained from nodules and rhizospheric soil belonged to genera Achromobacter,

Acinetobacter, Aeromonas, Bacillus, Bordetella, Bosea, Duganella, Ensifer,

Enterobacter, Klebsiella, Kocuria, Mesorhizobium, Microbacterium, Ochrobactrum,

Paenibacillus, Pseudomonas, Rhizobium and Serratia genera. Isolation of these genera

from nodules, rhizospheric soil of legumes and rhizospheric soil of non-legumes has

been frequently reported [10, 13, 14, 105, 114, 115, 117, 118].

Phosphorus is one of the major nutrients required by plants, being second only

to nitrogen. Most of the phosphorus in the soil is present in the form of insoluble

phosphates and cannot be utilized by the plants. The ability of bacteria to solubilize

mineral phosphates has been of interest to agricultural microbiologists as it can enhance

the availability of phosphorus and iron for plant growth. Similarly, IAA, a member of

the group of phytohormones, is generally considered to be the most important native

auxin. IAA may function as important signal molecule in the regulation of plant

development [164]. All isolates, except the Bosea spp. purified in the present study

showed IAA production in spent growth media and 34 isolates exhibited P-

solubilization in pure culture. Under field conditions, microorganisms with the P-

solubilization and IAA production abilities can play a significant role as PGPR when

applied as bio-fertilizer to different crops [117, 119-121]. In the present study, the effect

of incubation temperature on TCP-solubilization and IAA production by the bacterial

strains was also investigated. Incubation temperature of 30 oC was found to be the best

for P-solubilization and IAA production compared with 20 oC and 40 oC incubation

temperature. Tolerance of bacterial inoculants to high temperature is desirable and

important for their survival, growth and successful colonization under the conditions

similar to those found in the main chickpea growing area of Pakistan [119, 122]. All

strains performed well at 30 oC under controlled conditions and produced maximum

IAA and P-solubilization at this temperature. However, Ensifer sp. NFY8 and Serratia

4. Discussions

89

sp. 5D maintained their growth promoting traits at 40 oC. This temperature (40 oC) is

the maximum temperature recorded during chickpea cropping season in the main

chickpea growing area of the country.

Overall results of the field trials showed that chickpea inoculation with

symbiotic Mesorhizobium sp. along with Ensifer and Serratia spp. as free-living

bacterial isolates with phytohormones production and P-solubilization abilities

significantly improved plant growth and yield. This positive influence of bacterial

inoculation was consistent in all independent bacterial inoculation instances i.e.,

inoculation with all strains at different ecological sites and both chickpea varieties.

These results suggested that there is a great potential for the use of Ensifer sp.,

Mesorhizobium sp. and Serratia sp. as bacterial inocula for chickpea plants grown in

different ecological zones.

Comparison between the all-experimental sites selected for the field trials

showed that higher yield was obtained at NIBGE as compared to Thal dessert for all

treatments. This improved yield at NIBGE, Faisalabad experimental site could be due

to relatively high nutrient and moisture contents of the soil. The soil at this site was

irrigated at the time of sowing and also received about 40 % higher rainfall during the

experimental season than the Thal desert. Previously, Soltani et al. [123] have reported

about 90 % increase in yield of chickpea with full irrigation. Furthermore, several other

factors such a temperature, soil texture and organic matter content might also have

contributed to the observed difference in yield at two sites [47, 124-126].

The differences in the yield of both chickpea varieties were obvious at all sites

except AZRI, Bhakkar, regardless of inoculation or fertilization treatments. Chickpea

Desi-type varieties gave significantly higher grain and straw yield as compared to

Kabuli-type at both experimental sites for all treatments. This difference could be due

to its better genetic potential and ability to interact positively with the existing

environmental and biogeochemical factors. The variations in the yields of different

plant species grown under the same conditions have been reported previously [47]. The

positive influence of bacterial inoculation was consistent on both varieties indicating

that Ensifer spp., Mesorhizobium spp. and Serratia spp. can be used as an effective

single-strain inocula or as co-inoculants for both the chickpea varieties.

4. Discussions

90

In the present study, pyrosequencing of nifH gene directly amplified from

nodule DNA of chickpea revealed diversity of diazotrophic (nitrogen-fixing) bacteria

associated with chickpea. A significant fraction i.e., 88.83 % of the total sequences

retrieved from all sites belonged to genus Mesorhizobium and detected sequences were

identical to that found in some pure culture of mesorhizobia obtained from nodules in

the present study. Laranjo et al. [3] have described that only Mesorhizobium genus is

true endophyte of chickpea. This clear dominance of mesorhizobial nifH gene

sequences is due to the fact that Mesorhizobium is the only genus which can nodulate

the chickpea [3, 127-131]

Sequence analysis of nifH gene PCR-amplified from rhizospheric soil DNA

revealed diversity of diazotrophic (nitrogen-fixing) bacteria associated with chickpea.

A significant fraction i.e., 16.68 % of the total sequences retrieved from all sites

belonged to genus Mesorhizobium. In the present study diazotrophic genera like

Azospirillum, Azotobacter, Bradyrhizobium, Ensifer and Frankia were also detected

along with mesorhizobia both in the nodules as well as in rhizospheric soil samples.

Occurrence of the members of these genera has been frequently reported from different

crops [9, 107, 132, 133].

In the present study, pyrosequencing of 16S rRNA gene directly amplified from

nodule DNA of chickpea revealed enormous diversity of root nodule associated

bacteria. Legumes are known to harbor multiple endophytes in the nodules as

previously reported [10, 114]. Overall analysis of retrieved sequences revealed

dominance of taxa belonging to class α-Proteobacteria (56.99 %), followed by γ-

Proteobacteria (26.43 %), “uncultured” (7.67 %), Firmibacteria (4.93 %), β-


Dominance of Actinobacteria (26.55 %), β-Proteobacteria (18.09 %), α-

Proteobacteria (16.10 %) and γ-Proteobacteria (3.71 %) has also been reported in

studies conducted on maize rhizospheric soil [134]. Comparison of sequences retrieved

from different sites indicated that α-Proteobacterial sequences were less abundant

(15.29 %) in the nodules collected from Kallar Syedan (District Rawalpindi) as

compared to NIBGE experimental site (75.63 %). Class Firmibacteria were not

represented in the retrieved sequences from the site Kallar Syedan (District Rawalpindi)

and NIFA (District Peshawar). At Thal desert firmibacterial sequences were relatively

abundant compared with other sites. Firmibacteria are known to be resistant to extreme

4. Discussions

91

conditions similar to the weather conditions prevailing at Thal desert (e.g., high

temperature, nutrient deficient soil and desert) due to spore formation [135, 136]. At

the sites Kallar Syedan (District Rawalpindi) and Chowk Munda (District

Muzaffargarh), α-proteobacterial sequences were relatively under represented (i.e.,

15.29 % and 25.91 % of the total retrieved sequences, respectively) but at both sites

maximum number of γ-proteobacterial sequences (54.77 % and 52.14 %, respectively)

were detected. Comparison of the sequences retrieved from Desi- and Kabuli-type

chickpea at NIBGE site showed that α-proteobacterial sequences were relatively more

abundant in nodules of Desi-type than Kabuli-type. However, in the nodules of Kabuli-

type chickpea, actinobacterial, β-proteobacterial, γ-proteobacterial and firmibacterial

sequences were relatively more abundant compared to Desi-type chickpea. These

results suggest some preference of certain groups of bacteria to associate with a specific

chickpea host type, in addition to soil type and weather conditions. Our results also

support the recent findings by Bonito et al. [137] that variation in root fungal and

bacterial communities has relevance to plant host and microbial host preferences, as

well as to factors pertaining to soil conditions.

A significant fraction i.e., 52.77 % of the total 16S rRNA sequences recovered

from nodules belonged to genus Mesorhizobium. In the nodules of Desi-type chickpea

cultivated in experimental field NIBGE (Districted Faisalabad) sequences of the genus

Mesorhizobium were more abundant (71.98 % of the total sequences) as compared to

Kallar Syedan (District Rawalpindi) and Chowk Munda (District Muzaffargarh) where

the mesorhizobial sequences comprised about 12.49 % and 22.35 % of the total

sequences, respectively. Dominance of mesorhizobial sequences among the retrieved

sequences indicated that in the present study sequences of endophytes were preferably

amplified and detected. Similarly, it may be inferred that microbes detected in addition

to Mesorhizobium may also be endophytic. However, we cannot rule out PCR

amplifications of the sequences from any contaminating DNA or bacterial cells

escaping our nodule surface sterilization procedure.

In the present study about 29.22 % of the retrieved 16S rRNA sequences

originated from “uncultured” fraction of the bacterial populations associated with the

nodules. Percentage of “uncultured” bacterial sequences was higher in the nodules

collected from nutrient deficient soil at Chowk Munda (District Muzaffargarh). Similar

higher percentage (32 % to 43 %) of “uncultured” bacteria has been reported from the

4. Discussions

92

rhizospheric soil of Brazilian Cerrado which is a tropical ecosystem containing a

diverse mosaic of grassland, savanna, woodland and forest [138]. Future research will

clarify significance of these “uncultured” bacteria and their role in the rhizospheric soil

ecology.

The retrieved sequences from nodule DNA of chickpea revealed occurrence of

111 bacterial genera. Among these, isolation of 44 genera has been reported previously

from the nodules of different legumes [9-14]. Abundance of these 44 genera is reflected

by the fact that about 69.32 % of total sequences belonged to these bacteria and the

remaining 67 genera detected in the present study constituted only 1.46 % of total

sequences.

Sequence analysis of 16S rRNA gene PCR-amplified from chickpea

rhizospheric soil DNA revealed diversity of bacteria associated with chickpea. The

retrieved sequences belonged to diverse phyla including Proteobacteria (23.40 %),

Firmicutes (5.52 %), Actinobacteria (29.48 %), “uncultured” (28.04 %) and other

minor phyla (13.57 %). Further analysis of the retrieved sequences indicated that

retrieved sequences belonged to diverse taxa including α-Proteobacteria (10.44 %), γ-

Proteobacteria (3.49 %), “uncultured” (35.97 %), Firmibacteria (4.99 %), β-


Dominance of Actinobacteria (26.55 %), β-Proteobacteria (18.09 %), α-

Proteobacteria (16.10 %) and γ-Proteobacteria (3.71 %) has also been reported in

studies conducted on maize rhizospheric soil [134]. Analysis of the retrieved sequences

indicated that sequences of all the bacterial genera isolated in the present study were

retrieved along with numerous sequences belonging to diverse genera of culturable as

well as “uncultured” bacteria. A fraction i.e., 0.265 % of the total sequences retrieved

from all sites belonged to genus Mesorhizobium. In the present study 29.72 %

sequences belonging to 313 genera of culturable bacteria were retrieved along with

70.28 % sequences of “uncultured” bacteria.

Through retrieved 16S rRNA gene sequences, several genera of PGPR were

detected in the present study including several important PGPR like Actinomadura,

Agromyces, Aeromicrobium, Arthrobacter, Bacillus, Bosea, Burkholderia, Ensifer,

Flavobacterium, Kocuria, Lysinibacillus, Mesorhizobium, Microbacterium, Nocardia,

Paenibacillus, Pantoea, Pseudomonas, Rhizobium, Serratia, Sphingobacterium,

4. Discussions

93

Staphylococcus, Streptomyces and Yersinia that have been reported for phytohormone

production and phosphate solubilization [40, 139-145]. Among these PGPR, some

genera like Agromyces, Ancylobacter, Arthrobacter, Ensifer, Mesorhizobium,

Ochrobactrum, Rhizobium and Streptomyces have been reported as free-living or

symbiotic nitrogen fixers [115, 146-149].

High abundance of Serratia spp. affiliated 16S rRNA gene sequences in nodules

collected from the Thal desert was revealed and comprised up to 15 % of the total

retrieved sequences. This high abundance of Serratia spp. as chickpea endophytic

bacteria is likely to be beneficial because in healthy plants penetration into roots or

nodules by pathogenic bacteria is strictly controlled by the host plant defense system

and most likely only plant beneficial bacteria can be enriched by the rhizospheric soil

conditions or occupy endophytic niches. Their entrance in the plant systems is

compromised by the host plant because of their plant growth promoting attributes such

as phytohormone production, enhancing the availability of nutrients, and by acting as

bio-control agent to other plant pathogenic organisms [150-152]. This outcome

suggested a potential positive association of the organisms from this genus with

chickpea plants that was further explored in detail in this study by targeted isolation of

two Serratia strains and application as inocula for chickpea.

Comparison of the Serratia spp. affiliated endophytic microbial populations at

different sites indicated relative low abundance in the root nodules as compared to the

nodules (15 %) from the Thal desert area. This could be due to the differences in

geochemistry’s of sites. Soil in the Thal site has significantly lower concentrations of

K, total and available P as compared to the other sites. It is possible that under P-limited

conditions host plant selectively favored the organisms that have P-solubilization

ability [153]. This kind of selective enrichment or strong regulation of the endophytic

Rhizobium population in response to soil N content has been reported previously [154].

Previously, endophytic and phyllo epiphytic presence of Serratia spp. in Populus

deltoids and in spinach plants has been reported [155].

Among the retrieved Serratia spp. sequences through uncultured methods, 96

% sequences (1093) showed maximum similarity to Serratia marcescens and the

remaining 43 sequences were closely related to an “uncultured” Serratia species. This

suggested that among different Serratia spp., S. marcescens strains are predominant

4. Discussions

94

endophytes at our sites and secondly, sequences from a potential novel Serratia sp.

were present, although in relatively low abundance. To confirm potential role of the

genus Serratia on chickpea growth, isolation of two Serratia marcescens affiliated

strains were made from chickpea root nodules. Red coloration, a distinctive character

of Serratia colonies reported by Petersen and Tisa [156] was very helpful in the initial

screening and isolation. Both isolates, 5D and RTL100 were affiliated with Serratia

marcescens based on 16S rRNA gene sequencing and were closely related to the

dominant cluster detected through uncultured methods. Their high abundance in the

root nodules, detected through uncultured methods, most likely was the cause of

successful isolation from this particular group.

4.1 Conclusions

From the results obtained in the present study it can be concluded that mesorhizobial

strains are the main nodule endophytes as indicated by 16S rRNA and nifH gene

sequencing of the pure cultures as well as the retrieved sequences from nodule DNA.

A number of diverse bacterial taxa (111genera in nodules, 313 genera in rhizospheric

soil) were detected by culture-independent technique, which included sequences of

genera previously reported as nodulating or non-nodulating endophytes of other

legumes and several well-known PGPR. In the present study, Mesorhizobium sp. NTY7

co-inoculated with PGPR Ensifer sp., NFY8 and Serratia sp. 5D proved to be the most

effective inoculum for both chickpea varieties and the inocula may be used for

production of biofertilizer for chickpea. Future studies may focus on isolation of these

important “uncultured” PGPR genera using selective growth media and optimum

growth conditions and explored for their PGPR potential along with the symbiotic

mesorhizobia.

95

Appendices

Appendix A

Relative abundance of bacterial genera detected by 16S rRNA gene sequence

analysis in the root nodule of chickpea grown at different localities.


Genera detected L1 L2 L3 L4 L5 L6 L7 Acinetobacter 0.890 3.031 0.032 4.319 0.000 5.190 1.501

Actinomadura 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Actinophytocola 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Advenella 0.000 0.000 0.287 0.000 0.000 0.000 0.087

Aeromicrobium 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Agromyces 0.043 0.000 0.011 0.000 0.000 0.000 0.016

Amaricoccus 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Amycolatopsis 0.011 0.000 0.032 0.000 0.055 0.000 0.016

Ancylobacter 0.000 0.015 0.000 0.000 0.000 0.000 0.003

Arthrobacter 0.000 0.000 0.011 0.000 0.000 0.073 0.006

Bacillus 0.043 0.000 0.043 0.000 0.000 0.000 0.026

Bordetella 0.000 0.000 0.872 0.000 0.492 0.000 0.292

Bosea 0.011 0.015 0.032 0.042 0.000 0.000 0.019

Brevibacillus 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Brevundimonas 0.032 0.000 0.000 0.000 0.000 0.000 0.010

Burkholderia 0.000 0.000 0.000 4.906 0.000 0.000 0.375

Buttiauxella 0.172 0.000 0.128 0.084 0.000 0.439 0.115

Catellatospora 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Caulobacter 0.000 0.000 0.000 0.042 0.000 0.000 0.003

Cellvibrio 0.000 0.015 0.000 0.000 0.000 0.000 0.003

Chitinophaga 0.032 0.044 0.000 0.168 0.000 0.000 0.032

Chryseobacterium 0.011 0.000 0.000 0.168 0.109 0.000 0.022

Cohnella 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Comamonas 0.011 0.000 0.000 0.084 0.000 0.000 0.010

Cupriavidus 0.000 0.000 0.000 0.042 0.055 0.000 0.006

Delftia 0.000 0.000 0.000 1.593 0.000 0.000 0.122

Devosia 0.032 0.000 0.000 0.000 0.000 0.000 0.010

Dongia 0.043 0.044 0.000 0.000 0.000 0.000 0.022

Duganella 0.021 0.015 0.000 0.881 0.000 0.585 0.103

Dyadobacter 0.011 0.015 0.000 0.042 0.000 0.000 0.010

Dyella 0.000 0.000 0.000 0.000 0.055 0.000 0.003

Ensifer 0.568 0.350 0.043 0.000 0.000 0.658 0.289

Enterobacter 0.054 0.000 0.032 0.042 0.000 0.292 0.042

Filimonas 0.021 0.000 0.000 0.000 0.000 0.000 0.006

Flavobacterium 0.086 0.189 0.000 0.335 0.000 0.000 0.093

Gaiella 0.064 0.000 0.000 0.000 0.000 0.000 0.019

Gemmatimonas 0.000 0.015 0.000 0.000 0.000 0.000 0.003

Georgenia 0.000 0.000 0.032 0.000 0.000 0.000 0.010

Cont…

Appendices

96

Glycomyces 0.107 0.029 0.000 0.000 0.000 0.000 0.038

Gp3 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Gp4 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Herbaspirillum 0.000 0.000 0.000 0.964 0.000 0.000 0.074

Ilumatobacter 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Inquilinus 0.011 0.000 0.000 0.000 0.164 0.000 0.013

Isoptericola 0.011 0.029 0.000 0.000 0.000 0.000 0.010

Kocuria 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Kosakonia 0.021 0.000 0.000 0.000 0.000 0.000 0.006

Kribbella 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Labrys 0.011 0.000 0.021 0.084 0.000 0.000 0.016

Lechevalieria 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Luteimonas 0.000 0.015 0.011 0.000 0.000 0.000 0.006

Lysinibacillus 0.000 0.000 0.011 0.000 0.000 0.000 0.003

Lysobacter 0.032 0.015 0.000 0.000 0.000 0.000 0.013

Massilia 0.054 0.029 0.000 0.084 0.000 0.000 0.029

Mesorhizobium 56.01 71.98 52.46 12.49 22.35 47.37 52.77

Methylophilus 0.021 0.029 0.000 0.000 0.000 0.000 0.013

Methyloversatilis 0.000 0.015 0.000 0.000 0.000 0.000 0.003

Methylovorus 0.032 0.000 0.000 0.042 0.000 0.000 0.013

Microbacterium 0.021 0.015 0.011 0.000 0.055 0.000 0.016

Microvirga 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Moheibacter 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Mucilaginibacter 0.000 0.000 0.000 0.126 0.000 0.000 0.010

Mycobacterium 0.021 0.015 0.000 0.042 0.000 0.000 0.013

Myroides 0.021 0.000 0.000 0.000 0.000 0.000 0.006

Nitrososphaera 0.032 0.015 0.000 0.000 0.000 0.000 0.013

Nocardia 0.097 0.000 0.000 0.000 0.000 0.000 0.029

Nocardioides 0.054 0.015 0.000 0.000 0.000 0.000 0.019

Nocardiopsis 0.000 0.000 0.000 0.000 0.000 0.146 0.006

Nonomuraea 0.000 0.015 0.000 0.000 0.000 0.000 0.003

Novosphingobium 0.129 0.000 0.000 0.000 0.000 0.000 0.038

Ochrobactrum 0.075 0.015 0.159 0.000 0.219 0.000 0.087

Ohtaekwangia 0.064 0.015 0.000 0.000 0.000 0.000 0.022

Olivibacter 0.064 0.015 0.000 0.000 0.601 0.000 0.058

Opitutus 0.000 0.029 0.000 0.000 0.000 0.000 0.006

Oxobacter 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Paenibacillus 0.021 0.000 0.170 0.000 0.437 0.000 0.083

Pantoea 3.346 2.405 5.666 0.964 0.820 2.193 3.458

Phenylobacterium 0.011 0.000 0.000 0.000 0.000 0.000 0.003 Planococcaceae_incertae_sedis 0.000 0.000 6.293 0.000 0.000 0.000 1.899

Planomonospora 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Pontibacter 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Promicromonospora 0.204 0.073 0.011 0.000 0.109 0.000 0.087

Pseudomonas 4.719 2.507 0.149 28.72 0.109 5.994 4.475

Pseudorhodoferax 0.043 0.029 0.000 0.000 0.000 0.000 0.019

Pseudoxanthomonas 0.311 0.277 0.000 0.000 0.000 0.000 0.154

Psychrobacillus 0.000 0.000 0.011 0.000 0.000 0.000 0.003

Ralstonia 0.000 0.000 0.000 0.042 0.000 0.000 0.003

Rheinheimera 0.097 0.000 0.000 0.000 0.000 0.000 0.029

Rhizobium 1.373 0.583 0.106 0.252 0.109 0.804 0.632

Rhodobacter 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Roseomonas 0.000 0.000 0.000 0.000 0.055 0.000 0.003

Serratia 2.038 2.477 5.666 0.000 0.000 0.000 2.864

Cont…

Appendices

97

Shinella 0.011 0.015 0.000 0.000 0.000 0.000 0.006

Skermanella 0.043 0.000 0.000 0.000 0.000 0.000 0.013

Solirubrobacter 0.021 0.000 0.000 0.000 0.000 0.000 0.006

Sphaerobacter 0.000 0.015 0.000 0.000 0.000 0.000 0.003

Sphingobacterium 0.601 0.044 0.000 0.084 0.765 0.292 0.253

Sphingobium 0.021 0.000 0.000 0.000 0.000 0.000 0.006

Sphingomonas 0.064 0.029 0.000 0.000 0.000 0.000 0.026

Sporosarcina 0.000 0.000 0.128 0.000 0.000 0.000 0.038

Staphylococcus 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Stenotrophomonas 4.022 0.627 0.319 1.468 3.443 0.073 1.755

Steroidobacter 0.097 0.015 0.000 0.000 0.000 0.000 0.032

Streptomyces 0.225 0.044 0.011 0.000 0.000 0.000 0.080

Terrimonas 0.011 0.015 0.000 0.000 0.000 0.000 0.006

Tumebacillus 0.021 0.000 0.000 0.000 0.000 0.000 0.006

Variovorax 0.043 0.000 0.000 0.000 0.000 0.000 0.013

Xanthobacter 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Xanthomonas 0.064 0.073 0.000 0.000 0.000 0.000 0.035

Yersinia 0.000 0.000 0.000 0.210 0.000 0.000 0.016

Zavarzinella 0.011 0.000 0.000 0.000 0.000 0.000 0.003

Uncultured 23.316 14.748 27.246 41.677 70.000 35.892 27.313



Nodules

Appendix B

Bacterial genera detected by 16S rRNA gene sequence analysis in rhizospheric

soil of chickpea (Data shown in percentage)

Bacterial genera L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 Aciditerrimonas 0.038 0.036 0.053 0.015 0.010 0.033 0.030

Acidovorax 0.015 0.002

Acinetobacter 0.023 0.018 0.053 0.387 0.066 Acrocarpospora 0.010 0.002

Actinoallomurus 0.018 0.002

Actinocorallia 0.018 0.015 0.029 0.078 0.024 Actinokineospora 0.195 0.015 0.002

Actinomadura 0.184 0.048 0.114 0.179 0.068 0.134 0.121 0.113

Actinomycetospora 0.061 0.015 0.054 0.035 0.015 0.117 0.056 0.033 0.050 Actinophytocola 0.071 0.251 0.018 0.071 0.149 0.068 0.078 0.033 0.123

Actinoplanes 0.038 0.018 0.011 0.014

Actinopolymorpha 0.011 0.002 Adhaeribacter 0.225 0.024 0.114 1.297 0.071 0.060 0.078 0.134 0.132 0.229

Advenella 0.008 0.002

Aerococcus 0.015 0.002 Aeromicrobium 0.024 0.091 0.035 0.060 0.020 0.022 0.011 0.044

Agrococcus 0.018 0.002

Agromyces 0.262 0.205 0.054 0.035 0.045 0.088 0.145 0.121 0.113 Alterococcus 0.041 0.010 0.022 0.002

Amaricoccus 0.245 0.048 0.015 0.149 0.108 0.056 0.154 0.056

Aminobacter 0.071 0.008 Ammoniphilus 0.164 0.048 0.144 0.378 0.035 0.104 0.078 0.056 0.110 0.123

Amycolatopsis 0.024 0.616 0.198 0.549 0.283 0.117 0.011 0.011 0.309

Anaeromyxobacter 0.020 0.071 0.071 0.015 0.186 0.190 0.044 0.082 Aneurinibacillus 0.020 0.004

Aquabacterium 0.035 0.004 Aquicella 0.020 0.167 0.030 0.049 0.045 0.088 0.022

Arcicella 0.020 0.004

Arenimonas 0.119 0.177 0.089 0.010 0.055 0.034 Armatimonadetes_gp2 0.011 0.002

Armatimonadetes_gp4 0.020 0.190 0.304 0.162 0.496 0.194 0.352 0.357 0.099 0.314

Armatimonadetes_gp5 0.035 0.020 0.078 0.022 Armatimonas/Armatimonadetes_g

p1

0.048 0.008 0.018 0.053 0.029 0.033 0.022 0.022

Cont…

Appendices

98

Arthrobacter 0.204 0.190 3.277 15.45

9

1.205 0.522 0.421 0.603 0.198 2.964

Aspromonas 0.015 0.010 0.004

Aurantimonas 0.011 0.002

Azoarcus 0.048 0.030 0.117 0.134 0.022 0.052 Azohydromonas 0.030 0.180 0.035 0.075 0.039 0.112 0.070

Azomonas 0.011 0.002

Azonexus 0.010 0.011 0.004 Azospirillum 0.020 0.023 0.090 0.018 0.030 0.078 0.123 0.011 0.060

Azotobacter 0.008 0.018 0.089 0.059 0.045 0.034

Bacillariophyta 0.143 0.045 0.049 0.066 0.018 Bacillus 1.943 0.667 0.654 4.018 0.939 0.268 1.105 0.948 1.353 1.151

Bacteriovorax 0.011 0.002

Balneimonas 0.008 0.002 Bauldia 0.010 0.011 0.004

Blastocatella 0.008 0.030 0.078 0.056 0.032

Blastochloris 0.041 0.024 0.008 0.011 0.033 0.004 Blastococcus 0.368 0.167 0.471 0.595 0.284 0.134 0.098 0.112 0.275 0.279

Blastomonas 0.018 0.002

Blastopirellula 0.041 0.048 0.030 0.030 0.010 0.044 0.014 Bordetella 0.020 0.008 0.018 0.011 0.004

Bosea 0.020 0.122 0.018 0.045 0.059 0.022 0.011 0.056

Bradyrhizobium 0.061 0.036 0.319 0.075 0.068 0.045 0.088 BRC1_genera_incertae_sedis 0.020 0.015 0.036 0.015 0.020 0.022 0.011 0.018

Brevibacillus 0.119 0.030 0.018 0.053 0.030 0.039 0.067 0.055 0.040

Brevundimonas 0.020 0.018 0.015 0.010 0.011 0.006 Burkholderia 0.015 1.152 0.133

Byssovorax 0.018 0.015 0.020 0.033 0.014

Candidatus Hydrogenedens 0.010 0.002 Candidatus Koribacter 0.010 0.022 0.006

Catellatospora 0.048 0.035 0.030 0.020 0.078 0.022 0.026

Catelliglobosispora 0.036 0.004 Catenuloplanes 0.035 0.004

Caulobacter 0.018 0.104 0.016

Cellulomonas 0.018 0.010 0.011 0.006 Cellvibrio 0.024 0.045 0.039 0.022 0.011 0.018

Cesiribacter 0.010 0.022 0.006

Chelativorans 0.008 0.002 Chelatococcus 0.024 0.010 0.011 0.002

Chitinophaga 0.046 0.018 0.053 0.015 0.147 0.067 0.064

Chlorophyta 0.024 0.030 0.010 0.011 0.006 Chthonomonas/Armatimonadetes

_gp3

0.071 0.008 0.195 0.045 0.049 0.179 0.033 0.072

Clostridium III 0.095 0.015 0.098 0.045 0.044 0.030 Clostridium sensu stricto 0.020 0.008 0.018 0.060 0.117 0.112 0.011 0.056

Clostridium XI 0.011 0.002

Cohnella 0.020 0.024 0.015 0.090 0.030 0.020 0.022 0.022 0.026 Conexibacter 0.024 0.023 0.015 0.020 0.022 0.011 0.016

Corallococcus 0.015 0.004

Cryptosporangium 0.023 0.010 0.008 Cupriavidus 0.024 0.084 0.054 0.018 0.015 0.022 0.011 0.036

Cystobacter 0.020 0.048 0.084 0.036 0.018 0.045 0.039 0.033 0.042 Dactylosporangium 0.024 0.122 0.180 0.035 0.010 0.006

Dechloromonas 0.039 0.011 0.008

Defluviicoccus 0.015 0.002 Dehalococcoides 0.032

Deinococcus 0.020

Desertibacter 0.015 0.068 0.078 0.030 Desmospora 0.020 0.004

Desulfobulbus 0.008 0.002

Desulfocapsa 0.061 0.016 Desulfomonile 0.023 0.006

Desulfuromonas 0.038 0.010

Devosia 0.143 0.023 0.054 0.124 0.089 0.020 0.078 0.077 0.056 Domibacillus 0.061 0.018 0.029 0.024

Dongia 0.024 0.071 0.045 0.029 0.011 0.011 0.022

Duganella 0.301 0.030 0.020 0.042 Dyadobacter 0.053 0.030 0.020 0.011 0.016

Dyella 0.035 0.004

Ensifer 0.082 0.071 0.259 0.757 0.492 0.509 0.223 0.077 0.360 Euzebya 0.020 0.011 0.011 0.002

Falsibacillus 0.010 0.078 0.002

Flavisolibacter 0.030 0.126 0.035 0.075 0.029 0.056 Flavitalea 0.008 0.018 0.045 0.010

Flavobacterium 0.053 0.030 0.039 0.112 0.038

Cont…

Appendices

99

Flindersiella 0.020 0.071 0.008 0.015 0.011 0.044 0.006

Fluviicola 0.024 0.045 0.011 0.006

Fontibacillus 0.018 0.010 0.004

Gaiella 0.409 1.857 1.308 0.667 2.588 2.161 1.947 2.622 1.078 1.859

Geminicoccus 0.123 0.071 0.076 0.036 0.018 0.030 0.059 0.067 0.099 0.054 Gemmata 0.020 0.071 0.015 0.053 0.089 0.098 0.045 0.044 0.050

Gemmatimonas 1.329 0.976 0.403 0.198 0.479 0.715 0.489 0.580 1.166 0.480

Geobacillus 0.011 0.002 Geobacter 0.008 0.030 0.059 0.134 0.042

Geodermatophilus 0.307 0.852 0.270 0.248 0.104 0.029 0.179 0.165 0.332

Georgenia 0.041 0.008 0.010 0.033 0.022 0.010 Glycomyces 0.082 0.035 0.030 0.059 0.056 0.044 0.030

Gordonia 0.041 0.010 0.033 0.022 0.008

Gp1 0.018 0.029 0.011 0.010 Gp10 0.020 0.214 0.144 0.532 0.194 0.059 0.123 0.110 0.157

Gp17 0.143 0.053 0.053 0.015 0.108 0.033 0.066 0.050

Gp18 0.018 0.015 0.004 Gp2 0.010 0.011 0.004

Gp22 0.015 0.010 0.011 0.006

Gp25 0.020 0.048 0.068 0.036 0.124 0.030 0.068 0.201 0.033 0.090 Gp3 0.204 0.429 0.190 0.018 0.408 0.328 0.704 0.569 0.308 0.386

Gp4 0.833 0.243 0.144 1.010 0.700 0.499 0.580 0.385 0.492

Gp5 0.024 0.008 0.018 0.015 0.011 0.011 0.008 Gp6 0.450 3.429 1.331 0.577 1.329 2.086 1.682 2.064 1.826 1.551

Gp7 0.041 0.167 0.190 0.126 0.319 0.045 0.059 0.056 0.099 0.127

GpI 0.204 0.071 0.018 0.039 0.045 0.143 0.018 GpIV 0.011 0.002

Haliangium 0.010 0.002

Halobacillus 0.041 0.020 0.011 0.022 0.006 Halomonas 0.010 0.002

Herbaspirillum 0.018 0.002

Herpetosiphon 0.071 0.015 0.020 0.123 0.036 Hydrogenophaga 0.030 0.049 0.078 0.028

Hymenobacter 0.036 0.035 0.030 0.012

Hyphomicrobium 0.020 0.018 0.045 0.020 0.011 0.012 Iamia 0.038 0.011 0.012

Ideonella 0.018 0.002

Ignavibacterium 0.011 0.002 Ilumatobacter 0.082 0.119 0.030 0.036 0.124 0.179 0.157 0.145 0.099 0.107

Inhella 0.010 0.002

Inquilinus 0.137 0.177 0.060 0.029 0.011 0.072 Isoptericola 0.078 0.112 0.036

Jahnella 0.008 0.002

Janibacter 0.030 0.059 0.011 0.022 Janthinobacterium 0.015 0.004

Jiangella 0.020 0.024 0.015 0.015 0.039 0.056 0.022 0.024

Kibdelosporangium 0.008 0.002 Kineococcus 0.036 0.039 0.012

Kineosporia 0.018 0.002

Kocuria 0.102 0.076 0.631 0.176 0.067 0.055 0.137 Kofleria 0.008 0.011 0.004

Kribbella 0.095 0.646 0.126 0.390 0.179 0.049 0.011 0.044 0.263 Ktedonobacter 0.018 0.015 0.004

Labrys 0.023 0.071 0.030 0.018

Lacibacter 0.010 0.002 Latescibacteria_genera_incertae_

sedis

0.015 0.002

Lechevalieria 0.061 0.024 0.327 0.036 0.461 0.179 0.049 0.123 0.044 0.197 Legionella 0.008 0.035 0.015 0.020 0.022 0.016

Lentzea 0.106 0.075 0.022

Litorilinea 0.061 0.071 0.023 0.018 0.018 0.068 0.078 0.066 0.038 Luteimonas 0.020 0.018 0.045 0.010 0.045 0.011 0.018

Lysinibacillus 0.061 0.008 0.011 0.033 0.004

Lysobacter 0.061 0.071 0.053 0.072 0.266 0.164 0.176 0.156 0.066 0.137 Marmoricola 0.061 0.048 0.061 0.072 0.177 0.149 0.117 0.056 0.055 0.098

Massilia 0.048 0.236 0.757 0.603 0.075 0.108 0.022 0.022 0.249

Mesorhizobium 0.041 0.071 0.122 0.162 0.815 0.328 0.147 0.279 0.055 0.265 Methanomassiliicoccus 0.008 0.054 0.010 0.022 0.014

Methanoregula 0.008 0.002

Methanosarcina 0.023 0.006 Methanospirillum 0.015 0.004

Methylobacillus 0.018 0.002

Methylobacterium 0.015 0.036 0.177 0.015 0.010 0.032 Methylocaldum 0.018 0.002

Methylophilus 0.041 0.008 0.035 0.060 0.011 0.022 0.016

Cont…

Appendices

100

Methylovorus 0.035 0.238 0.020 0.040

Microbacterium 0.020 0.095 0.175 0.216 0.035 0.045 0.020 0.033 0.055 0.090

Microbulbifer 0.010 0.002

Microlunatus 0.041 0.095 0.015 0.036 0.160 0.134 0.059 0.078 0.066 0.070

Micromonospora 0.061 0.024 0.030 0.071 0.030 0.049 0.089 0.044 0.046 Microvirga 0.348 0.357 1.589 1.477 0.603 0.462 0.626 0.591 0.352 0.941

Modestobacter 0.036 0.004

Mucilaginibacter 0.886 0.100 Mycobacterium 0.082 0.119 0.182 0.054 0.213 0.298 0.137 0.156 0.099 0.173

Myxococcus 0.041 0.024 0.114 0.018 0.015 0.049 0.022 0.033 0.048

Nakamurella 0.035 0.004 Nannocystis 0.024 0.046 0.054 0.018 0.020 0.033 0.011 0.030

Natronococcus 0.011 0.002

Naxibacter 0.015 0.054 0.010 Niabella 0.018 0.002

Niastella 0.024 0.036 0.089 0.015 0.010 0.011 0.018

Nitriliruptor 0.368 0.020 0.011 0.198 0.006 Nitrobacter 0.010 0.002

Nitrosomonas 0.020 0.022 0.008

Nitrososphaera 0.654 4.048 0.852 0.378 0.975 1.818 1.682 1.852 2.222 1.290 Nitrosospira 0.082 0.714 0.030 0.018 0.071 0.015 0.010 0.045 0.044 0.030

Nitrospira 0.184 0.327 0.288 0.284 0.387 0.470 0.469 0.429 0.380

Nocardia 0.143 0.143 0.099 0.378 0.071 0.104 0.059 0.067 0.143 0.113 Nocardioides 0.818 0.262 2.585 1.982 1.081 1.818 0.616 0.658 0.561 1.503

Nocardiopsis 0.024 0.008 0.089 0.010 0.011 0.011 0.018

Nonomuraea 0.020 0.048 0.114 0.018 0.124 0.045 0.196 0.156 0.033 0.119 Noviherbaspirillum 0.015 0.108 0.035 0.030 0.010 0.011 0.028

Novosphingobium 0.102 0.054 0.142 0.373 0.022 0.055 0.076

Ochrobactrum 0.102 0.024 0.220 1.207 0.142 0.164 0.127 0.167 0.066 0.285 Ohtaekwangia 0.245 0.405 0.182 0.180 0.142 0.492 0.577 0.513 0.319 0.358

Olivibacter 0.030 0.010 0.006

Opitutus 0.020 0.095 0.038 0.072 0.461 0.268 0.196 0.100 0.055 0.163 Ornithinimicrobium 0.307 0.054 0.010 0.067 0.165 0.020

Oscillochloris 0.204 0.010 0.022 0.006

Oxalophagus 0.015 0.054 0.010 Oxobacter 0.022 0.004

Paenibacillus 0.071 0.167 0.523 0.124 0.075 0.274 0.179 0.143 0.213

Panacagrimonas 0.010 0.011 0.004 Pantoea 0.072 0.035 0.134 0.049 0.040

Paracoccus 0.011 0.002

Pedobacter 0.048 0.018 0.035 0.030 0.010 0.022 0.012 Pedomicrobium 0.095 0.023 0.018 0.035 0.253 0.029 0.033 0.044 0.058

Pelagibacterium 0.011 0.002

Pelomonas 0.160 0.018 Pelotomaculum 0.008 0.002

Peredibacter 0.011 0.002

Phaselicystis 0.008 0.002 Phenylobacterium 0.041 0.071 0.160 0.108 0.425 0.104 0.108 0.067 0.055 0.149

Phycicoccus 0.018 0.015 0.004

Pirellula 0.123 0.167 0.129 0.054 0.053 0.268 0.342 0.257 0.143 0.197 Planctomyces 0.286 0.143 0.046 0.030 0.029 0.078 0.220 0.036

Planifilum 0.020 0.004 Planobispora 0.008 0.002

Planococcaceae_incertae_sedis 0.035 0.004

Planomicrobium 0.010 0.002 Planomonospora 0.024 0.020 0.022 0.011 0.008

Polaromonas 0.035 0.004

Pontibacter 4.438 0.071 0.023 0.072 0.060 0.176 0.357 2.420 0.121 Porphyrobacter 0.010 0.002

Promicromonospora 0.020 0.601 0.313 0.421 0.078 0.011 0.299

Prosthecomicrobium 0.015 0.002 Pseudokineococcus 0.020 0.004

Pseudolabrys 0.020 0.024 0.015 0.018 0.015 0.008

Pseudomonas 0.184 0.167 0.018 0.266 0.939 0.978 0.268 0.022 0.404 Pseudonocardia 0.722 0.144 0.408 0.566 0.235 0.268 0.176 0.422

Pseudorhodoferax 0.060 0.008

Pseudoxanthomonas 0.018 0.358 0.470 0.100 0.163 Psychrobacter 0.015 0.002

Ramlibacter 0.008 0.018 0.018 0.010 0.008

Rathayibacter 0.015 0.002 Rhizobacter 0.024 0.018 0.011 0.002

Rhizobium 0.018 0.337 1.043 0.704 0.100 0.340

Rhodococcus 0.061 0.008 0.015 0.020 0.045 0.033 0.016 Rhodocytophaga 0.108 0.015 0.117 0.045 0.046

Rhodoplanes 0.015 0.004

Cont…

Appendices

101

Roseimicrobium 0.010 0.002

Roseomonas 0.023 0.018 0.029 0.045 0.022

Rubellimicrobium 0.048 0.152 0.396 0.035 0.209 0.098 0.179 0.022 0.167

Rubrobacter 0.041 0.095 0.144 0.072 0.248 0.030 0.303 0.257 0.066 0.185

Saccharibacteria_genera_incertae_sedis

0.038 0.018 0.045 0.020 0.011 0.024

Saccharomonospora 0.023 0.010 0.002

Saccharopolyspora 0.018 0.071 0.015 0.029 0.022 0.022 Saccharothrix 0.020 0.053 0.104 0.049 0.045 0.011 0.044

Salinibacter 0.015 0.002

Segetibacter 0.053 0.006 Serratia 0.033 0.006

Shimazuella 0.091 0.015 0.029 0.011 0.034

Shinella 0.018 0.045 0.008 Sideroxydans 0.008 0.002

Singulisphaera 0.035 0.004

Sinomonas 0.018 0.002 Skermanella 0.041 0.167 0.335 0.396 0.106 0.164 0.293 0.234 0.099 0.267

Solimonas 0.008 0.018 0.004

Solirubrobacter 0.777 0.690 1.080 0.216 1.631 0.700 0.851 1.094 0.737 0.951 Sorangium 0.023 0.020 0.011 0.012

Sphaerisporangium 0.082 0.024 0.053 0.015 0.010 0.011 0.011 0.012

Sphaerobacter 0.048 0.030 0.108 0.030 0.020 0.022 0.066 0.032 Sphingobacterium 0.030 0.010 0.045 0.014

Sphingobium 0.015 0.020 0.006

Sphingomonas 0.015 0.018 0.124 0.060 0.010 0.011 0.032 Sphingopyxis 0.015 0.010 0.004

Spirillospora 0.020 0.020 0.011 0.011 0.006

Sporacetigenium 0.030 0.029 0.045 0.018 Sporichthya 0.024 0.068 0.018 0.033 0.011 0.026 Sporolactobacillaceae_incertae_sedis 0.102 0.018 0.055 0.002

Sporomusa 0.039 0.008 Sporosarcina 0.204 0.071 0.008 0.036 0.071 0.020 0.033 0.143 0.024

Stenotrophomonas 0.368 0.333 0.418 3.640 0.549 0.447 0.430 0.424 0.352 0.796

Steroidobacter 0.368 0.643 0.456 0.378 0.354 0.581 0.293 0.346 0.495 0.400 Streptomyces 0.307 0.524 1.946 2.360 0.868 0.894 0.538 0.647 0.407 1.212

Streptophyta 0.030 0.847 0.035 0.060 0.059 0.112 0.145

Streptosporangium 0.035 0.004 Subdivision3_genera_incertae_sedis 0.020 0.048 0.053 0.060 0.039 0.056 0.033 0.032

Sulfuricurvum 0.008 0.002

Sulfuritalea 0.023 0.006 Syntrophobacter 0.020 0.039 0.045 0.011 0.016

Terrabacter 0.142 0.030 0.010 0.022

Terriglobus 0.018 0.002 Terrimonas 0.008 0.071 0.015 0.010 0.011 0.016

Thermoactinomyces 0.048 0.010 0.022 0.002

Thermocatellispora 0.010 0.002 Thiobacillus 0.144 0.038

Truepera 0.041 0.015 0.162 0.022 0.022

Tumebacillus 0.123 0.137 0.126 0.089 0.015 0.205 0.156 0.066 0.131 Turicibacter 0.018 0.002

Vampirovibrio 0.015 0.011 0.004 Variovorax 0.024 0.152 0.054 0.071 0.045 0.020 0.011 0.011 0.066

Vasilyevaea 0.024 0.068 0.018 0.060 0.010 0.011 0.011 0.032

Virgisporangium 0.020 0.048 0.010 0.022 0.033 0.006 WPS-1_genera_incertae_sedis 0.038 0.036 0.142 0.045 0.108 0.056 0.068

WPS-2_genera_incertae_sedis 0.190 0.091 0.018 0.213 0.104 0.068 0.056 0.088 0.088

Xanthomonas 0.119 0.016 Zavarzinella 0.167 0.220 0.018 0.106 0.209 0.254 0.179 0.077 0.183

Uncultured 80.45

0

77.28

6

71.06

1

54.81

1

68.66

4

71.58

0

74.10

7

74.38

1

78.98

8

70.27

8




rhizospheric soil

102

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