RABIA KHALID 03-arid-80prr.hec.gov.pk/jspui/bitstream/123456789/6868/1/... · ISOLATION, IDENTIFICATION AND CHARACTERIZATION OF GROUNDNUT RHIZOBACTERIA TO IMPROVE CROP Department

ISOLATION, IDENTIFICATION AND CHARACTERIZATION

OF GROUNDNUT RHIZOBACTERIA TO IMPROVE CROP

Department of Soil Science & Soil and Water

Faculty of Crop and Food Sciences

Arid Agriculture University Rawalpindi

ii



YIELD

RABIA KHALID

03-arid-80

Department of Soil Science & Soil and Water Conservation


Pir Mehr Ali Shah


Pakistan

2015



Conservation

iii



YIELD

by

RABIA KHALID

(03-arid-80)

A thesis submitted in partial fulfillment of

the requirements for the degree of

Doctor of Philosophy

in

Soil Science

Department of Soil Science & Soil and Water Conservation


Pir Mehr Ali Shah


Pakistan

2015

iv

CERTIFICATION

I hereby undertake that this research is an original one and no part of this

thesis falls under plagiarism. If found otherwise, at any stage, I will be responsible

for the consequences.

Student’s Name: Rabia Khalid Signature: ____________

Registration No: 03-arid-80 Date: ________________

Certified that contents and form of the thesis entitled “Isolation,

Identification and Characterization of Groundnut Rhizobacteria to Improve

Crop Yield” submitted by Ms. Rabia Khalid have been found satisfactory for the

requirement of the degree.

Supervisor: ________________________ (Prof. Dr. Safdar Ali)

Member: __________________________ (Dr. Rifat Hayat)

Member: __________________________ (Dr. Ghulam Shabbir) Chairperson: _____________________

Dean: ____________________________

Director, Advanced Studies: ___________________________

v

vi

CONTENTS

Page

List of Tables viii

List of Figures ix

ACKNOWLEDGMENT xi

ABSTRACT xii

1. INTRODUCTION 1

2. REVIEW OF LITERATURE 6

2.1 IDENTIFICATION AND CHARACTERIZATION OF PGPR 7

2.1.1 Taxonomy of PGPR 7

2.1.1.1 Phenotypic characterization 9

2.1.1.2 Chemotaxonomic characterization 11

2.1.1.3 Genotypic characterization 12

2.1.2 Taxonomy of Rhizobia 14

2.2 PLANT GROWTH PROMOTING RHIZOBACTERIA 23

2.2.1 Mechanism of PGP by PGPR 24

2.2.1.1 Phosphorus solubilization 24

2.2.1.2 Phytohormone production 31

2.2.1.3 Nitrogen fixation 36

2.2.1.3.1 Symbiotic nitrogen fixing bacteria 37

2.2.1.3.2 Non-symbiotic nitrogen fixing bacteria 38

2.3 APPLICATION OF PGPR AS BIOFERTILIZER 41

3. MATERIALS AND METHODS 43

3.1 SURVEY FOR SAMPLES COLLECTION 43

3.2 ISOLATION AND PURIFICATION OF BACTERIA 44

vii

3.2.1 Isolation and Purification of Rhizobacteria from Soil 44

3.2.2 Isolation and Purification of Bacteria from Nodules 44

3.3 MAINTANCE OF BACTERIAL CULTURE 45

3.4 PLANT GROWTH PROMOTING CHARACTERIZATION 45

3.4.1 Phosphate Solubilization 45

3.4.2 Synthesis of Indole-3-Acetic Acid (IAA) 48

3.4.3 Amplification of nifH gene 50

3.5 BIOCHEMICAL AND PHENOTYPIC CHARACTERIZATION 50

3.6 IDENTIFICATION USING MOLECULAR TECHNIQUE 52

3.6.1 Preparation of DNA template 52

3.6.2 PCR Amplification of 16S rRNA and Housekeeping Genes 52

3.6.3 Sequence Analysis 53

3.6.4 Phylogenetic Analysis (Bioinformatics) 58

3.7 EVALUATION UNDER POT AND FIELD CONDITIONS 58

3.8 PLANT ANALYSIS 61

3.9 ASSESSMENT OF N2-FIXATION BY NATURAL 15N 61

3.10 SOIL ANALYSIS 62

3.11 VALIDATION OF NOVEL BACTERIAL SPECIES 62

3.11.1 Morphology and Phenotypic Characterization 63

3.11.2 DNA Template for 16S rRNA and Housekeeping genes 64

3.11.3 Extraction of Bacterial DNA for DDH and G+C Contents 64

3.11.3.1 DNA-DNA hybridization 65

3.11.3.2 Determination of G+C contents 66

3.11.4 Fatty Acid Profiling 67

3.12 STATISTICAL ANALYSIS 68

viii

4. RESULTS AND DISCUSSION 69

4.1 CHARACTERIZATION FOR PGP TRAITS 69

4.1.1 Phosphate Solubilizations 70

4.1.2 Indole-3-Acetic Acid (IAA) Synthesis 71

4.1.3 nifH gene Amplification 72

4.1.4 Gram staining, Catalase and Oxidase Activity 77

4.2 MOLECULAR IDENTIFICATION OF BACTERIA 78

4.2.1 16S rRNA Gene Sequencing 78

4.2.2 Housekeeping Genes Sequencing 79

4.2.3 Phylogenetic Analysis of 16S rRNA and Housekeeping Genes 83

4.3 BIOCHEMICAL AND PHENOTYPIC ANALYSIS 84

4.4 BIOCHEMICAL CHARACTERIZATION OF PGPRs 109

4.5 GREEN HOUSE EXPERIMENT 114

4.5.1 Inoculation Effect of PGPRs on Growth Parameters 114

4.5.2 Inoculation Effect of selected PGPRs on N2-Fixation 119

4.6 FIELD EXPERIMENT 122

4.6.1 Inoculation Effect with PGPRs on Growth Parameters 122

4.6.2 Inoculation Effect with PGPRs on N2-Fixation 128

4.7 VALIDATION OF NOVEL SPECIES BN-19T 131

4.7.1 Morphology and Phenotypic Characterization 132

4.7.2 Phylogenetic Analysis of 16S rRNA and Housekeeping Genes 133

4.7.3 DDH and Chemotaxonomic Analysis 138

SUMMARY 146

LITERATURE CITED 151

APPENDICES 203

ix

List of Tables

Table No. Page

1. An overview of Rhizobium sp. in six genera 15

2. Beneficial effects of PGPR inoculation on various crops 25

3. Composition of media used for bacterial isolation 47

4. Composition of Nautiyal media 49

5. Primers used for PCR amplification and sequencing 54

6. Composition of PCR mixture 55

7. Physicochemical characteristics of AAUR and Koont soils 59

8. Plant growth promoting traits of isolated strains 73

9. Identification of bacterial strains by 16S rRNA gene sequencing 80

10. Sequence similarity and accession numbers of housekeeping gene 85

11. Biolog of Rhizobium strains isolated from groundnut nodules 105

12. Antibiotic resistance test of isolated Rhizobium strains 108

13. Biochemical characterization of selected PGPRs by API 20E 110

14. Biochemical characterization of selected PGPRs by API ZYM 112

15. PGP characterization of bacterial strains used in pot experiment 113`

16. Effect of inoculation of PGPR on growth parameters of groundnut 115

17. Effect of inoculation of PGPR on N2-fixation of groundnut 120

18. Percent increase in yield and groundnut in pot experiment 123

19. Effect on yield of groundnut as affected by inoculation of PGPRs 125

20. Effect PGPR inoculation on N2-fixation of groundnut 129

21. Differential features of Rhizobium pakistanensis sp. nov. 135

22. Sequence similarities for 16S rRNA housekeeping genes and DDH 143

23. Cellular fatty acid contents of BN-19T and reference strains 145

x

List of Figures

Fig. No. Page

1. Schematic diagram of mode of action of PGPR 8

2. Different compounds released by PSB for P solubilization 30

3. Schematic diagram for bacterial isolation 46

4. PCR cycling conditions for nifH gene amplification 51

5. PCR cycling conditions for 16S rRNA and atpD gene 56

6. PCR cycling conditions for recA and glnII gene amplification 57

7. Phylogenetic tree of BN2, 10, 14, 17, 26, 28, 49, GS-1 and GS-5 86

8. Phylogenetic tree of BN-3 constructed by neighbor-joining method 87

9. Phylogenetic tree of BN-4, 31, 32, 43, 44, 46, 47 and 55 88

10. Phylogenetic tree of BN-5 belonging to genus Arthrobacter 89

11. Phylogenetic tree of Rhizobium sp. BN-6, 12, 19, 20, 23, 39 and 50 90

12. Phylogenetic tree of BN-8, BN-24 and GS-6 91

13. Phylogenetic tree of BN-16 belonging to genus Curtobacterium 92

14. Phylogenetic tree of BN-22 and GS-7 belonging to Acinetobacter 93

15. Phylogenetic tree of BN-29 and BN-30 94

16. Phylogenetic tree of BN-29 belonging to genus Pantoea 95

17. Phylogenetic tree of BN-29 belonging to genus Erwinia 96



20. Phylogenetic tree of GS-3 belonging to genus Chryseobacterium. 99

21. Phylogenetic tree of GS-2 belonging to genus Kocuria. 100

22. Phylogenetic tree of Rhizobium strains for housekeeping gene atpD 101

23. Phylogenetic tree of Rhizobium strains for housekeeping gene recA 102

xi

24. Phylogenetic tree of Rhizobium strains for housekeeping gene glnII 103

25. Scanning electron microscopic picture of R. pakistanensis BN-19T 134

26. Phylogenetic tree of R. pakistanensis BN-19T based on 16S rRNA gene 139

27. Phylogenetic tree of R. pakistanensis BN-19T based on atpD gene 140

28. Phylogenetic tree of R. pakistanensis BN-19T based on recA gene 141

29. Phylogenetic tree of R. pakistanensis BN-19T based on glnII gene 142

xii

ACKNOWLEDGEMENTS

I wish to thank Almighty Allah for blessing me with strength and

perseverance to complete this study. I offer my humble gratitude to the Holy

Prophet Hazrat Muhammad (SAW) who is forever a torch of guidance for the

humanity as a whole.

I pay my thanks and respect to my supervisors, Prof. Dr. Safdar Ali for his

support in each and every step of Ph.D. and Dr. Rifat Hayat who helped me in

planning and executing this study and guided me with valuable suggestions to

make my academic goals attainable. I am also very thankful to Dr. Ghulam

Shabbir, for his guidance during the study. I wish to extend special thanks to Prof.

Dr. Wen Xin Chen and Dr. Xin Hua Sui, Rhizobium Research Centre, China

Agricultural University, Beijing, China for helping me to have an in-depth

understanding of soil microbiology and description of novel taxa. I wish to express

gratitude to Higher Education Commission of Pakistan for financing this study

through Ph.D. Indigenous Scholarship scheme.

I am very much indebted to my respected parents for their love and support

throughout my life and to my brothers and sister for their prayers and

encouragement. Heartiest thanks to my fellow Ph.D. students and especially to my

best friend Ummay Amara for their friendly cooperation and moral support. The

patience and support provided by my husband and daughter during my Ph.D. will

always be remembered by me.

(Rabia Khalid)

xiii

ABSTRACT

Plant growth promoting rhizobacteria (PGPR) have attained global

recognition as biofertilizers owing to their role in facilitating plant growth and

development in sustainable agricultural system. Groundnut (Arachis hypogea L.) is

high value cash crop in rainfed Pothwar, northern Punjab, Pakistan, yielding 85%

of the country’s total groundnut. The national average yield (1.1 t ha-1)is less than

the potential yield (4 t ha-1) and can be increased by inoculating groundnut with its

specific symbiont. The present study was designed with the objectives to isolate

bacterial strains from groundnut nodules and rhizospheric soil tocharacterize and

identify potential bacterial strains. The most potential bacterial strains were

evaluated under controlled and field conditions to confirm their growth promoting

effect on growth and N2-fixation of groundnut. A novel Rhizobium sp. BN-19T

from groundnut nodules was also validated following minimal standards of

bacterial identification. For bacterial isolation and identification, extensive survey

was carried out in Pothwar (Distt. Attock and Chakwal) for collection of groundnut

nodules and rhizospheric soil.Around 75 bacterial strains of different genera were

isolated and designated as BN-1, BN-2, BN-3…. (from nodules) and GS-1 to GS-

34 (from rhizospheric soil). These isolated bacterial strains were characterized for

plant growth promoting (PGP) traits like nifH gene amplification, indole acetic acid

(IAA) production, P solubilization, catalase and oxidase production.Biochemical

characterization was done by using API 20E kit and BIOLOG GN-2 microplate

reader. Resistance of Rhizobium sp. to seven antibiotics at 4 different levels (µg

mL-1): 5, 50, 100, 300 was tested. All the strains solubilized mineral phosphate

between30 to 700µg mL-1. The production of IAA was in the range of 10-92 µg

mL-1 (with tryptophan). For identification, 16S rRNA gene sequences of the strains

xiv

were obtained by PCR amplification using universal primers. For strains identified

as Rhizobiumhousekeeping genes [atpD (510 bp), recA (530 bp) and glnII (600bp)]

sequencing was also performed. The consensus sequences were BLAST using

EzBiocloud server database and retrieved using bioinformatics softwares i.e.

Bioedit, Clustal X and Mega 5 to construct neighbor-joining phylogenetic trees.

Seven most promising strains along with Rhizobium sp.were selected on the basis

of PGP traits and inoculated to groundnut under controlled conditions. Three

strains(BN-55, GS-4 and GS-6) enhanced yield under controlled conditions as

compared to other treatments, hence proved to be promising among seven tested

isolates. These strains were further tested in field along with full (N-P @ 20-40 kg

ha-1) and half recommended doze (N-P @ 10-20 kg ha-1) of chemical fertilizers, to

confirm their beneficial effect on groundnut yield and N2-fixation. The co-

inoculation of bacterial strains along with chemical fertilizer improved yield (76%)

and N2-fixation (193%) under field conditions. Phylogenetic analysis based on 16S

rRNA gene sequence revealed that one Rhizobium species BN-19T, isolated from

groundnut nodules exhibited sequence similarity of 97.4% with closely related

strains hence indicating its position as a distinct species in genus Rhizobium.

Sequence analysis of housekeeping genes (with sequence similarities of ≤92%) and

DNA-DNA relatedness between the strain BN-19T and reference strains (less than

30%), further confirmed that BN-19T represented a novel Rhizobium species, for

which the name Rhizobium pakistanensis sp. nov. is proposed. Our study confirmed

that soil has a very large community of bacteria having PGP characteristics, which

when applied as biofertilizer to crops improve nutrient use efficiency, yield and N2-

fixation. Farmers of the rainfed tract could raise their profitability by co-inoculation

of PGPR and chemical fertilizer.

1

Chapter 1

INTRODUCTION

Biological Nitrogen Fixation (BNF) is enzymatic conversion of

unavailable molecular nitrogen (~79% N2) into ammonium through nitrogenase

enzyme released by microorganisms thus utilizable by plants. These

microorganisms are referred as nitrogen fixers or diazotrophs (Hayat et al., 2010).

The beneficial bacteria residing in rhizosphere, interacting with plant roots and

have positive influence on plant growth are termed as plant growth promoting

rhizobacteria (PGPR) or plant health promoting rhizobacteria (PHPR).PGPR

enhance plant growth and development by many ways inducing their beneficial

impact by direct and indirect mechanisms (Ahemad and Kibret, 2014). The

different mechanisms of plant growth promotion by PGPR include fixing of

atmospheric nitrogen and make it available to the plants, synthesize antibiotics,

bacterial and fungal antagonistic substances and siderophores which convert

insoluble iron into soluble form, release different plant growth regulators,

particularly auxins and gibberellins, solubilze minerals like phosphorus and

increase nutrient cycling and synthesize plant growth stimulating enzymes

(Nihorimbereet al., 2011). A diversified range of bacteria belonging to genera

Pseudomonas, Azospirillum, Azotobacter, Bacillus, Klebsiella, Enterobacter,

Rhizobium, Xanthomonas and Serratia turned out to be effective PGPR by

promoting plant growth (Ahemad and Kibret, 2014).

Groundnut (Arachis hypogaea L.) is a leguminous crop and sandy loam

soils are ideal for obtaining best pod yield. The key groundnut producing areas of

Pakistan include Chakwal, Attock and Rawalpindi in Punjab, Karak and Sawabi in

1

2

NWFP and Sanghar in Sindh province. It is a high value cash crop of resource poor

farmers in rainfed Pothwar (district Chakwal and Attock), northern Punjab,

Pakistan, yielding 85% of the country’s total groundnut. Different varieties of

spreading and erect groundnut grown on an area of 1 million hectare with an

average yield of 1.1 t ha-1 as compared to 4 t ha-1 average yield of USA and China

(Khalid et al., 2015).The major constraints in the groundnut yield are low input

application to the crop by the growers, low nutrient availability in soil,

unavailability of high yielding adopted varieties, unpredictable environmental

conditions, unavailability of biofertilizer for groundnut in the country and

no/inefficient specific rhizobial inoculation. The nodulation of groundnut could be

possible by the indigenous Rhizobia present in the soil however; inoculation of

groundnut seeds with specific Rhizobium would be helpful in attaining better yields

(El-Akhal et al., 2008).Research regarding groundnut nodulating rhizobia

groundnut are rareand encompass the ones carried out in Argentina (Taurian et al.,

2006; Bogino et al., 2006), South Africa (Law et al., 2007) and China (Zhang et al.,

1999).Therefore, there is a need to identify efficient rhizobium strains to be used as

groundnut inoculum.

The genus Rhizobium has an ability of nitrogen-fixation in leguminous

plants. Literature has also confirmed the capacity of some PGPRs to modify nodule

formation or even biological nitrogen fixation when they are co-inoculated with

Rhizobium (Qureshiet al., 2009; Sánchezet al., 2014). The co-inoculation of

effective Rhizobium sp. by PGPR to enhance N2-fixation by legumes is an approach

to increase N2 supply in eco-friendly agricultural production systems. As a legume,

groundnut is also considered as an efficient N2-fixer, fixes about 100-130 kg N ha-1

3

y-1 yet there is potential to increase nitrogen fixation by manipulation of rhizobial

strains (Latif et al., 2009). It is reported that only a fraction of total agricultural

need of nitrogen comes from natural or synthetic fertilizers, the remainder is largely

satisfied through biological nitrogen fixation of atmospheric nitrogen (Robertson

and Vitousek, 2009). Inoculation with Bradyrhizobium strain of groundnut has been

reported by many research workers for increasing pod yield. The beneficial effects

of inoculum in improving groundnut yield were reported by Badawi et al. (2011).

Similarly, Miah and Karim (1995) recorded 32% increased grain yield of groundnut

with inoculum application.

Various morphological and phenotypic methodologies are being used to

identify and characterize bacteria. Early bacterial classifications were based mainly

on the morphological characteristics, but prokaryotes lack the morphological,

developmental and fossil evidence to track their phylogenetic relationships. To

overcome this intrinsic weakness, molecular and biochemical characteristics (e.g.,

on the peptidoglycan structure of cell wall, cellular fatty acid and isoprenoid

quinine) were used extensively for classification and identification of bacterial

strains, and this approach produced more phenotypic information now helpful in

bacterial taxonomy. The development of molecular biological techniques such as

DNA sequencing, DNA G+C contents determination and DNA-DNA hybridization,

RAPD, AFLP, PCR fingerprinting, housekeeping genes analysis (Oren and Garrity,

2013) and the improvement of tools of chemical analysis have dramatically

changed bacterial determinative taxonomy towards bacterial phylogenetics and

resulted in identification of bacteria at the sub-species level. The small subunit

ribosomal RNA (16S rRNA) is highly conserved molecule and universally exists

4

for every bacterial species. For this reason the comparative analysis of 16S rRNA

gene sequence has been recognized as the most powerful method in molecular

phylogenetics of bacteria (Woese, 1992; Kim and Chun, 2014). For the purpose of

phylogenetic analysis, genes used as molecular taxonomic markers should be

universal and not show any mutation to specific conditions (Patwardhan et al.,

2014), and in this regard housekeeping genes are most suitable and useful for strain

discrimination and identification of bacteria (Janda and Abbott, 2007).

The overall goals of this study were to validate novel bacteria from

groundnut nodule and access the co-inoculated effects of potential PGPR and

Rhizobium strains along different combinations of N and P fertilizers on N2-fixation

and yield of groundnut under rainfed conditions of Pothwar. Considering the

significance of co-inoculation and fertilizer management, the study was planned

with the following objectives:

1. Isolation and characterization of bacterial strains from groundnut nodules

and rhizospheric soil for growth promoting traits.

2. Identification of potential bacterial strains using molecular technique i.e.

16S rRNA and housekeeping (atpD, recA and glnII,) gene sequencing

techniques.

3. Evaluating effectiveness of co-inoculation of most potential PGPR and

Rhizobium along with inorganic fertilizers to improve growth and yield of

groundnut in pot and field experiments.

5

4. Validation of novel bacterial strain by adopting minimal standards (DNA-

DNA hybridization, fatty acid profiling, BIOLOG) for bacterial

identification.

6

Chapter 2

REVIEW OF LITERATURE

Numerous biotic and abiotic factors are responsible for influencing plant

growth and development in soil. The narrow layer of soil associated with plant

roots described as “rhizosphere” is exceptionally significant and active region for

root functions and metabolic processes. For the first time, the term rhizosphere was

used by Hiltner (1904), for describing the soil thin layer encompassing the roots,

wherein the microbial communities are prompted by the functions of roots. The

rhizosphere comprises of a vast range of microorganisms including bacteria, fungi,

algae and protozoa. It is assumed that rhizosphere population is dominated by

bacteria, therefore they are influencing the plant growth and development most

significantly, particularly by taking into consideration their compatibility for root

colonization (Antoun and Kloepper, 2001; Barriuso et al., 2008).

The bacteria which reside in the rhizosphere can be categorized on the basis

of their impact on plant growth and the manner in which they are associated with

roots: some are pathogens; on the other hand others induce favorable impacts.

Rhizobacteria colonizing rhizospheric soil and exerting beneficial influence varying

from direct mode of actions to an indirect impression on plant physiology are

referred to be plant growth promoting rhizobacteria (PGPR) (Ahemad andKibret,

2014). From the last few decades, the significance of rhizosphere has increased

significantly because of the effective attributes of the biosphere; as a result, there is

a tremendous increase in the identified number of PGPR. Different bacterial genera

including Pseudomonas, Azospirillum, Azotobacter, Klebsiella, Enterobacter,

Alcaligenes, Arthrobacter, Burkholderia, Rhizobium, Bacillus and Serratia are

6

7

revealed to promote plant development (Glick, 1995; Hayat et al., 2010). Several

PGPR inoculants are available commercially that have the potential for promoting

plant development by no less than single mode of action e.g. inhibition of disease

occurrence (bioprotectants), enhance nutrients availability and uptake

(biofertilizers), and/or synthesis of phytohormones (biostimulants) (Figure 1).

2.1 IDENTIFICATION AND CHARACTERIZATION OF PGPR FOR

AGRICULTURE

Identification and characterization procedures are very important to describe

the taxonomic status of bacteria. These characteristic include morphological,

phenotypical and molecular characterization primarily determined by fatty acid

profiling, determination of G+C contents [mol (%)], DNA–DNA hybridization

(DDH), 16S rRNA and housekeeping genes sequence analysis.

2.1.1 Taxonomy of PGPR

The term taxonomy describes the associations between similar species as

well as the categorization, identification and nomenclature of organisms (Coenye et

al., 2005). Generally taxonomy is considered an alternative of biosystematics or

systematic and conventionally it is alienated into three steps: (i) categorization,

designating as the systematic placement of moicrorganisms into taxonomic clusters

based on level of similarity among them; (ii) nomenclature i.e., naming the

organisms and (iii) identifying bacteria by establishing procedures to confirm if the

bacteria is associated with already defined nomenclature system or is a new species

(Chun and Rainey, 2014). The bacterial species is defined by full genomic DDH

values by exhibiting greater than 70% DNA-DNA relatedness value with 5°C

8

Figure 1:Schematic diagram demonstrating the mode of action of PGPR to support plant growth.

8

9

°C less ∆Tm (Janda and Abbott, 2007). Even though many latest characterization

techniques have been formulated over the last decades, the standard procedure of

identification is still unchanged. The recent techniques for identification of different

bacterial species consisted of four categories based on (i) conventional

morphological, physiological and biochemical characterization, (ii) biochemical

tests by using rapid mini kits (i.e., API kits, VITEK cards, or Biolog plates), (iii)

chemotaxonomic characterization (like polyacrylamide gel electrophoresis [PAGE],

or fatty acid methyl ester [FAME] profiles) (4) genotypic characterization (16S

rRNA, housekeeping gene analysis and DDH). From 1950s, the phenotypic

approaches for bacterial identification proved to be inappropriate and insufficient.

To overcome this gap, nucleic acids studies and the chemotaxonomic analyses were

investigated. Nevertheless, it is not possible to establish standardized circumstances

to obtain bacterial growth by chemotaxonomic analysis; therefore polyphasic

procedure has become mandatory in taxonomic categorization till species level

(Figueiredo et al., 2010). Polyphasic technique pertains the incorporation of

genotypic, chemotaxonomic, and phenotypic insights together so that the authentic

grouping of an organism could be performed (Schleifer, 2009). The distinct

practices applied for polyphasic description of rhizobacteria are explained below.

2.1.1.1 Phenotypic characterization

Phenotype encompasses morphological, biological, and biochemical

attributes of an organism (de Vos et al., 2009). The conventional phenotypic

investigations comprised of colony morphology (appearance, size, shape and edges)

and microscopic study of bacteria (shape, endospore, flagella), growth pattern of

the microbe on various media, growth spectrum of bacteria on variable

10

concentrations of NaCl, pH, and temperature, and resistance towards specific

antibiotics, etc. Despite the fact that cell wall structure is investigated, still the

Gram staining assay is an effective criterion for bacterial identification.

Biochemical diagnostic protocols used for bacterial species identification consist of

their association with fermentation reactions, carbohydrates and nitrogen metabolic

activity and their aerobic or non-aerobic nature (Heritage et al., 1996; Rodríguez-

Díaz et al., 2008). A major drawback related to phenotypic characterization is that

the reproducibility of information obtained from phenotypic tests amongst different

labs is not consistent, and only standardized protocol should be followed to perform

the analysis. Other negative impact of phenotypic procedures included uncertain

manifestations of characters by different genes, wherein the similar bacteria would

possibly exhibit distinctive phenotypic traits in varying environments. Hence it is

necessary that phenotypic results should be accessed by using same database of

type strains of closely associated species.

Biochemical kits comprising of dehydrated reagents are available for

traditional biochemical tests in order to confirm taxonomic status of bacteria. The

biological reaction (growth, synthesis of enzymes, etc.) is initiated by the addition

of standardized inoculum and the results are interpreted according to

manufacturer’s instructions. The phenotypic kit API 50CH comprised of one

negative control and 49 different carbohydrates and is used for the identification of

Bacillus and Paenibacillus species, whereas API 20E kits have resulted in the

correct identification of Pseudomonas strains (Boyd et al., 2005). Biolog is

regarded as quick and comparatively easy assay of bacterial identification

11

(Tshikhudo et al., 2013). It comprised of utilization of 95 carbon sources and one

blank control.

2.1.1.2 Chemotaxonomic characterization

The chemotaxonomic techniques used for PGPR identification consist of

fatty acid analysis, cellular protein contents determination by PAGE, polar lipid

tests, quinone determination, composition of cell wall, pyrolysis mass spectrometry,

Fourier transformed infrared spectroscopy, Raman spectroscopy, and matrix-

assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass

spectrometry. Fatty acids are considered as the main components of lipids and

lipopolysaccharides and are employed for taxonomic identification. At present, it is

the only chemotaxonomic approach which is associated with the professional

database for identification purpose. Fatty acid profiles exhibiting differences in

chain length, double-bond position, and substituent groups are appropriate for

classification of unknown bacterial species and also for comparative study of

profiles attained under same lab environment (Suzuki et al., 1993).

Standardized growth conditions and procedure for analysis is requisite for

sodium dodecyl sulfate-PAGE of whole-cell proteins. It has made a worthwhile

addition to polyphasic taxonomic analysis of aerobic endospore forming bacteria

(Logan et al., 2009). The composition of cell wall is determined in Gram-positive

bacteria containing differential peptidoglycan in their cell wall constitution. The

Gram-negative bacteria cell wall has uniform peptidoglycan composition hence

providing minor structural insights (Silhavy et al., 2010). Isoprenoid quinones is an

important component of cytoplasm of most prokaryotes performing essential

12

function in electron transport, active transport and oxidative phosphorylation

(Nowicka and Kurk, 2010). Although the majority of the polar lipids detected in

bacteria are not structurally characterized, still they are used as an important

taxonomic marker for identification of novel bacteria. Similarly, quinines (MK-7,

MK-8, and MK-9) are documented to represent the family Bacillaceae (Logan et

al., 2009).

2.1.1.3 Genotypic characterization

Genotypic approach is the one that is oriented towards DNA or RNA

molecules. The methods dealing the taxonomy at molecular level have completely

transformed the microbial identification and nomenclature system. Various

methods like restriction fragment length polymorphism (RFLP), plasmid profiling,

ribotyping, amplified ribosomal DNA restriction analysis (ARDRA), pulsed field

gel electrophoresis (PFGE), and randomly amplified polymorphic DNA (RAPD)

are now available to differentiate bacteria up to species level (Monteiro et al. 2009).

Determination of guanine plus cytosine (G+C) contents is considered

amongst the traditional genotypic approaches. For already identified and validated

species, its range is not >3%, and for identified genus the range is not >10%

(Fournier et al., 2006). The procedure of DNA-DNA hybridization (DDH) is

oriented on the principle i.e., DNA is denatured at elevated temperatures, but

lowering down the temperature resulted in bringing back the molecule to its native

form (reassociation). This process compares the full genome of two bacterial strains

(Goris et al., 2007). Generally for a bacterial species to consider as strain, it must

exhibit DDH value >70%. Mora (2006) has compared different methods for DDH,

13

but all the methods give the relative percentage of similarity between two bacterial

species but do not give the actual gene sequence result. To cope with the drawbacks

of DDH, DNA microarray method was developed, which make use of fragmented

DNA rather than whole genomic DNA.

Undoubtedly, the introduction of rRNA molecule to compare the

evolutionary linkages among species has revolutionized the taxonomy. During the

last few decades, it is approved that bacterial classification should complement the

innate inter-relationship between bacteria which is possible due to phylogenetic

linkages as determined by 16S or 23S rRNA sequencing (Vos et al., 2012). In cell

rRNA molecules exist in three forms, i.e., 5S, 16S, and 23S and they could be

employed in phylogenetic studies; however 16S rRNA molecule (1500 bp) stands

out as the universal marker. 16S rRNA gene depicts conserved traits, it lacks

horizontal transfer between species, and its evolution rate represents variant

hierarchy among prokaryotes (Woo et al., 2008). It consists of conserved and

variable portions, and conserved region is deployed to design universal primers for

16S rRNA gene amplification, whereas for the study of comparative taxonomy

variable region is coded. The 16S rRNA gene sequence is deposited in GenBank

databases including DDBJ (http://www.ddbj.nig.ac.jp/) and

NCBI(http://www.ncbi.nlm.nih.gov/). Sequences (fasta files) of closely associated

bacterial strains could be obtained from DDBJ, NCBI or EzBiocloud for

constructing phylogenetic trees and computing pair wise distances, wherein the

degree of relatedness among closely related strains could be analysed. This

evolutionary tree confirms the genus of the bacterial strain and exhibit its

relationship with its closely related species, the strains in the same cluster or

14

revealing >97% 16S rRNA gene sequence similarity, are acquired from different

culture collections so that genotypic, chemotaxonomic, and phenotypic analysis

could be performed. Recently, by association with experimental data obtained by

comparing total genomic DNA (DDH), it is recommended that a resemblance less

than 98.7-99% of 16S rRNA gene sequences between two bacterial candidates is

satisfactory to reckon them as distinct species. In such circumstances, DNA–DNA

hybridization should be conducted and the strains exhibiting DDH relatedness less

than 70% are regarded as distinct species (Goris et al., 2007). Eventually, the

housekeeping or protein coding genes sequences e.g. glnII, dnaK, gyrB, rpoB, recA,

atpD etc. have also conserved nature and have the potential to be used in taxonomic

study to differentiate species. Clustering patterns for rpoB sequence when

compared with 16S rRNA sequences of Paenibacillus proved to be similar, hence

authenticating the potential of housekeeping genes for species identification (Mota

et al., 2005). Wang et al. (2007a) incorporated gyrB gene sequence in the analysis

of B. subtilis and Cerritos et al. (2008) used recA sequence for Bacillus sp.

identification which resulted in the proposal of novel Bacillus species.

2.1.2 Taxonomy of Rhizobia

Generally the word rhizobia depicts the bacteria forming nodules on roots

and stems and exist in endo-symbiosis with legumes, belonging to α-proteobacteria

group of family Rhizobiaceae, consist of genus Allorhizobium (de Lajudie et al.,

1998a), Azorhizobium (Dreyfus et al., 1988), Bradyrhizobium (Jordan, 1982),

Rhizobium (Frank, 1879), Mesorhizobium (Jarvis et al., 1997), and Sinorhizobium

(Chenet al., 1988) (Table 1). Other N2-fixing proteobacteria belong to

15

genusAzoarcusare also described which are in association with grasses(Franche et

al.,

16

Table 1:A timeline overview of Rhizobium species in six genera along with their source of isolation.

Name Type Strain Year of isolation

Source of isolation Nodulation Reference

Allorhizobium A. undicola ORS 992 1998 Neptunia natans + de Lajudie et al., 1998a Azorhizobium A. caulinodans ORS 571 1988 Sesbania rostrata + Dreyfus et al., 1988 A. doebereinerae UFLA1-100 2006 Sesbania virgata + Moreira et al., 2006 A. oxalatiphilum NS12 2013 Rumex sp. - Lang et al., 2013 Bradyrhizobium B. japonicum ATCC 10324 1982 Cicer arietinum + Jordan, 1982 B. denitrificans ATCC 43295 1986 Aeschynomene indica + Hirsch and Müller, 1985 B. elkanii USDA 76 1993 Glycine max + Kuykendall et al., 1992 B. liaoningense 2281 1995 Glycine soja, Glycine max + Xu et al., 1995 B. yuanmingense CCBAU 10071 2002 Lespedeza spp. + Yao et al., 2002 B. betae PL7HG1 2004 Beta vulgaris - Rivas et al., 2004 B. canariense BTA-1 2005 Chamaecytisus proliferus + Vinuesa et al., 2005 B. jicamae PAC68 2009 Pachyrhizus erosus + Ramírez-Bahena et al.,

2009 B. pachyrhizi PAC48 2009 Pachyrhizus erosus + Ramírez-Bahena et al.,

2009 B. iriomotense EK05 2010 Entada koshunensis + Islam et al., 2010 B. cytisi CTAW11 2011 Cytisus villosus + Chahbourne et al., 2011 B. lablabi CCBAU 23086 2011 Lablab purpureus, Arachis hypogaea + Chang et al., 2011

17

B. huanghuaihaiense CCBAU 23303 2012 Glycine max + Zhang et al., 2012a B. daqingense CCBAU 15774 2013 Glycine max + Wang et al., 2013a B. diazoefficiens USDA 110 2013 Glycine max + Delamuta et al., 2013 B. oligotrophicum ATCC 43045 1985 Rice - Ramírez-Bahena et

al., 2013 B. ganzhouense RITF806 2014 Acacia melanoxylon + Lu et al., 2014 B. icense LMTR 13 2014 Phaseolus lunatus + Durán et al., 2014 B. ingae BR 10250 2014 Inga laurina + Da Silva et al., 2014 B. manausense BR 3351 2014 Vigna unguiculata + Silva et al. 2014 B. neotropicale BR 10247 2014 Centrolobium paraense + Zilli et al., 2014 B. ottawaense OO99 2014 Glycine max + Yu et al., 2014 B. paxllaeri LMTR 21 2014 Phaseolus lunatus + Durán et al., 2014 B. retamae Ro19 2014 Retama sphaerocarpa + Guerrouj et al., 2014 B. rifense CTAW71 2014 Cytisus villosus + Chahboune et al., 2014 Mesorhizobium M. loti ATCC 700743 1982 Lotus corniculatus + Jarvis et al., 1997 M. mediterraneum UPM-Ca36 1995 Cicer arietinum + Jarvis et al., 1997 M. huakuii ATCC 51122 1991 Astragalus sinicus + Jarvis et al., 1997 M. ciceri UPM-Ca7 1991 Cicer arietinum + Jarvis et al., 1997 M. tianshanense A-1BS 1995 Saline desert soil + Jarvis et al., 1997 M. plurifarium CIP 105884 1998 Acacia species + de Lajudie et al., 1998b M. amorphae ACCC 19665 1999 Amorpha fruticosa + Wang et al., 1999 M. chacoense CECT 5336 2001 Prosopis alba + Velázquez et al., 2001 M. septentrionale SDW014 2004 Astragalus adsurgens + Gao et al., 2004 M. temperatum SDW018 2004 Astragalus adsurgens + Gao et al., 2004

18

M. thiogangeticum SJT 2006 Clitoria ternatea + Ghosh and Roy, 2006 M. albiziae CCBAU 61158 2007 Albizia kalkora + Wang et al., 2007b M. caraganae CCBAU 11299 2008 Caragana spp. + Guan et al., 2008 M. gobiense CCBAU 83330 2008 Desert soil + Han et al., 2008a M. tarimense CCBAU 83306 2008 Lotus frondosus + Han et al., 2008a M. australicum WSM2073 2009 Biserrula pelecinus + Nandasena et al., 2009 M. metallidurans STM 2683 2009 Anthyllis vulneraria + Vidal et al., 2009 M. opportunistum WSM2075 2009 Biserrula pelecinus + Nandasena et al., 2009 M. shangrilense CCBAU 65327 2009 Caragana species + Lu et al., 2009a M. alhagi CCNWXJ12-2 2010 Alhagi sparsifolia + Chen et al., 2010 M. robiniae CNWYC 115 2010 Robinia pseudoacacia + Zhou et al., 2010 M. camelthorni CCNWXJ 40-4 2011 Alhagi sparsifolia + Chen etal., 2011 M. muleiense CCBAU 83963 2012 Cicer arietinum Zhang et al., 2012 M. silamurunense CCBAU 01550 2012 Astragalus species + Zhao et al., 2012 M. tamadayense Ala-3 2012 Anagyris latifolia, Lotus berthelotii. + Ramírez-Bahena et

al., 2012 M. abyssinicae AC98c 2013 Acacia abyssinica - Degefu et al., 2013 M.hawassense AC99b 2013 Acacia abyssinica, Acacia tortilis - Degefu et al., 2013 M. qingshengii CCBAU 3460 2013 Astragalus sinicus + Zheng et al., 2013 M. sangaii SCAU7 2013 Astragalus luteolus + Zhou et al., 2013 M. shonense AC39a 2013 Acacia abyssinica - Degefu et al., 2013 Rhizobium R. leguminosarum USDA 2370 1879 Pisum sativum + Frank, 1879 R. phaseoli ATCC 14482 1926 Phaseolus vulgaris + Dangeard, 1926 R. trifolii ATCC 14480 1926 Pisum sativum + Dangeard, 1926

19

R. lupini ATCC 10319 1886 Lupinus spp. + Eckhardt et al., 1931 R. fredii PRC 205 1984 Glycine max + Scholla and Elkan, 1984 R. galegae ATCC 43677 1989 Galega orientalis, Galega oficinalis + Lindström, 1989 R. huakuii 103 1991 Astragalus sinicus + Chen et al., 1991 R. tropici CIAT 899 1991 Phaseolus vulgaris + Martínez-Romero et al.,

1991 R. etli CFN 42 1993 Phaseolus vulgaris + Segovia et al., 1993 R. gallicum R602sp 1997 Phaseolus vulgaris + Amarger et al., 1997 R. giardinii H152 1997 Phaseolus vulgaris + Amarger et al., 1997 R. hainanense CCBAU 57015 1997 Desmodium sinuatum + Chen et al., 1997 R. huautlense SO2 1998 Sesbania herbacea + Wang et al., 1998 R. mongolense ATCC BAA-116 1998 Medicago ruthenica + van Berkum et al., 1998 R. vitis K309 1990 Vitis spp - Young et al., 2001 R. undicola ORS 992 1998 Neptunia natans - Young et al., 2001 R. radiobacter ATCC 19358 1902 Soil - Young et al., 2001 R. rhizogenes ATCC 11325 1930 Soil - Young et al., 2001 R. rubi CFBP 5509 1940 Rubus spp. - Young et al., 2001 R. yanglingense SH 22623 2001 Gueldenstaedtia multiflora + Tanet al.,2001 R. indigoferae AS 1.3054 2002 Indigofera amblyantha + Wei et al., 2002 R. sullae IS123 2002 Hedysarum coronarium L. + Squartini et al., 2002 R. loessense AS1.3401 2003 Astragalus complanatus + Wei et al., 2003 R. larrymoorei ATCC 51759 2001 Ficus benjamina - Young, 2004 R. daejeonense L61 2005 Cyanide-degrading bioreactor - Quan et al., 2005 R. lusitanum P1-7 2006 Phaseolus vulgaris + Valverde et al., 2006 R. cellulosilyticum ALA10B2T 2007 Sawdust of Populus alba - García-Fraile et al., 2007

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R. fabae CCBAU 33202 2008 Viciafaba + Tian et al., 2008 R. miluonense CCBAU 41251 2008 Lespedeza chinensis + Gu et al., 2008 R. multihospitium CCBAU 83401 2008 Halimodendron halodendron + Han et al., 2008b R. oryzae Alt 505 2008 Oryza alta + Peng et al., 2008 R. pisi DSM 30132 2008 Pisum sativum + Ramírez-Bahena et al.,

2008 R. selenitireducens B1 2008 Bioreacter - Hunter et al., 2008 R. alamii GBV016 2009 Medicago ruthenica - Berge et al., 2009 R. alkalisoli CCBAU 01393 2009 Caragana intermedia + Lu et al., 2009b R. mesosinicum CCBAU 25010 2009 Albizia, Kummerowia, Dalbergia + Lin et al., 2009 R. tibeticum CCBAU 85039 2009 Trigonella archiducis-nicolai + Hou et al., 2009 R. soli DS-42 2010 soil in Korea - Yoon et al., 2010 R. aggregatum ATCC 43293 1986 Lake water + Kaur et al., 2011 R. borbori DN316 2011 Textile wastewater sludge - Zhang etal., 2011a R.endophyticum CCGE 2052 2011 Phaseolus vulgaris - López-López etal., 2011 R.herbae CCBAU 3011 2011 Astragalus membranaceus + Ren etal., 2011a R.pseudoryzae J3-A127 2011 Rice - Zhang etal., 2011b R.pusense NRCPB10 2011 Chickpea rhizospheric soil - Panday etal., 2011 R.rosettiformans W3 2011 Hexachlorocyclohexane dump site - Kaur etal., 2011 R.tubonense CCBAU 85046 2011 Oxytropis glabra + Zhang etal., 2011c R.vallis CCBAU 65647 2011 Phaseolus vulgaris + Wang etal., 2011 R.vignae CCBAU05176 2011 Vigna radiata + Ren etal., 2011b R.grahamii CCGE 502 2012 Dalea leporina, Clitoria ternatea + López-López etal., 2012 R.halophytocola YC6881 2012 Rosa rugosa - Bibi etal., 2012 R.leucaenae CENA 183 2012 Leucaena leucocephala + Ribeiro etal., 2012

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R. mesoamericanum CCGE 501 2012 Phaseolus vulgaris, Mimosa pudica + López-López etal., 2012 R.nepotum 39/7 2012 Prunus cerasifera - Pulawska etal., 2012a R.petrolearium SL-1 2012 Oil contaminated soil - Zhang etal., 2012b R.skierniewicense Ch11 2012 Chrysanthemum, cherry plum - Puławska etal., 2012b R.sphaerophysae CCNWGS0238 2012 Sphaerophysa salsula + Xu etal., 2012 R.taibaishanense CCNWSX0483 2012 Kummerowia striata + Yao etal., 2012 R. calliandrae CCGE524 2013 Calliandra grandiflora + Rincón-Rosales et

al., 2013 R. freirei PRF 81 2013 Phaseolus vulgaris + Dall'Agnol et al., 2013 R. jaguaris CCGE525 2013 Calliandra grandiflora + Rincón-Rosales et

al., 2013 R.mayense CCGE526 2013 Calliandra grandiflora + Rincón-Rosales et

al., 2013 R. paknamense L6-8 2013 Lemna aequinoctialis - Kittiwongwattana and

Thawai, 2013 R. subbaraonis JC85 2013 Beach sand - Ramana et al., 2013 R. tarimense PL-41 2013 Soil - Turdahon et al., 2013 R. azibense 23C2 2014 Phaseolus vulgaris + Mnasri et al., 2014 R. endolithicum JC140 2014 Beach sand samples - Parag et al., 2014 R. flavum YW14 2014 Triazophos applied soil - Gu et al., 2014 R. laguerreae FB206 2014 Vicia faba + Saïdi et al., 2014 R. lemnae L6-16 2014 Lemna aequinoctialis - Kittiwongwattana and

Thawai, 2014 R. paranaense PRF 35 2014 Phaseolus vulgaris + Dall’Agnol et al., 2014 R. populi K-38 2014 Populus euphratica - Rozahon et al., 2014

22

R. straminoryzae CC-LY845 2014 Rice straw - Lin et al., 2014 R. smilacinae PTYR-5 2015 Smilacina japonica - Zhang et al., 2015 Sinorhizobium S. fredii PRC 205 1984 Glycine soja + Chen et al., 1988 S. xinjiangense CCBAU 110 1988 Glycine max + Chen et al., 1988 S. meliloti ATCC 9930 1926 Medicago sativa + De Lajudie et al., 1994 S. saheli ATCC 51690 1994 Sesbenia cannabina + De Lajudie et al., 1994 S. terangae ATCC 51692 1994 Acacia laeta + De Lajudie et al., 1994 S. medicae A 321 1996 Medicago truncatula + Rome et al., 1996 S. arboris TTR 38 1999 Prosopis chilensis + Nick et al., 1999 S. kostiense TTR 15 1999 Acacia senegal + Nick et al., 1999 S. kummerowiae AS 1.3046 2002 Kummerowia stipulacea + Wei et al., 2002 S. morelense Lc04 2002 Leucaena leucocephala + Wang et al., 2002 S. americanum CFNEI 156 2004 Acacia species + Toledo et al., 2004

23

2009), and nodule formation by Buckholderia sp which belong to β-

proteobacteriasubclass revealed that there is diverse symbiotic association of

bacteria with legumes (Moulin et al., 2001). The rhizobia are unique in their

potential to synthesize Nod factors (Hirsch et al., 2001), for their synthesis,

nodulating genes nodABCD and host specific genes (hsn) are responsible. The

nodulation genes and other nif genes are present on Sym plasmids rather than on

chromosomes, which carries housekeeping genes (Barnett et al., 2001). Frank

(1879) for the first time named the bacterium Rhizobium leguminosarum isolated

from chick pea nodules, however Fred et al. (1932) was the first one to establish the

taxonomic diversity of rhizobia and categorized them on growth basis, but

nowadays polyphasic approach is implemented for rhizobial classification (de

Lajudie et al., 1994; Jarvis et al., 1997). These studies have resulted in validation of

many new species of Rhizobium Chen et al., 1997; van Berkum et al., 1998), and

till date one hundred and fifty species in six genera have been identified and

validated (http://www.bacterio.net/), however this total figure depicting rhizobium

species possibly equalize the overall number of legume species (approximately

18,000). Nearly all rhizobium have the potential to nodulate multiple host species

indicating their genetic diversity,however several rhizobium are isolated from a

single legume species. The studies on groundnut reported that it mostly form

effective nodules with slow-growing rhizobia belonging to genus Bradyrhizobium,

such as the species B. japonicum,B. elkanii,B. lablabi,B. yuanmingense and B.

iriomotense (Chang et al., 2011; El-Akhal et al., 2008; Muñoz et al., 2011; Wang et

al., 2013), but rarely with fast-growing Rhizobium species (Khalid et al., 2015; El-

Akhal et al., 2008; El-Akhal et al., 2009; Taurian et al., 2006). Santos et al. (2007)

revealed the dominance of fast-growing rhizobia which form effective nodules on

24

groundnut in Northeastern Brazil soils. Similarly during present study, Khalid et al.

(2015) isolated fast-growing rhizobia species namely R. alkalisoli, R. loessense, R.

huautlense, R. herbae and R. pusense from groundnut root nodules grown in

Pothwar, Pakistan, along with a potential novel Rhizobium candidate validated as R.

pakistanensis.

2.2 PLANT GROWTH PROMOTING RHIZOBACTERIA (PGPR)

The bacteria exerting positive effects on plant growth (PGPR) have been

recognized to possess some basic intrinsic characteristics: (i) PGPR should inhabit

the soil zone surrounding the roots (ii) they should thrive, multiply and survive in

competition with soil microorganisms, and (iii) they must endorse plant growth and

development (Kloepper, 1994). In many studies, a single PGPR revealed to perform

multiple functions (Kloepper, 2003; Vessey, 2003). Gray and Smith (2005)have

categorized PGPR into extracellular (ePGPR), prevailing in the soil surrounding the

roots and residing inside the epidermis, and intracellular (iPGPR), present in the

internal cells of the roots inside the nodules (Figueiredo et al., 2011). PGPR are

grouped on the basis of the functions they performed by Somers et al. (2004) as (i)

biofertilizers (increasing nutrients availability to the plants), (ii) phytostimulators

(synthesize phytohormones), (iii) bioremediators (degradation of organic

pollutants) and (iv) biopesticides (preventing diseases, mostly by the antibiotics and

antifungal metabolites synthesis). Various examples of ePGPR are Agrobacterium,

Arthrobacter, Azotobacter, Azospirillum, Bacillus, Burkholderia, Caulobacter,

Chromobacterium, Erwinia, Flavobacterium, Micrococcous, Pseudomonas and

Serratia etc. The example if iPGPR are the bacteria belonging to genus Rhizobium

(Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, Sinorhizobium

25

and Rhizobium) of the family Rhizobiaceae (Bhattacharyya and Jha, 2012). PGPR

belonging to genus Micromonospora sp., Streptomyces sp., Streptosporangium sp.,

and Thermobifida sp. served to be potential biocontrol agents against fungal

pathogens of roots (Franco-Correa et al., 2010).

2.2.1 MechanismofPlant Growth Promotion by PGPR

The plant metabolism is accelerated by PGPR through the production of

various substances like plant growth regulators, siderophores, solubilization of

insoluble phosphates and nitrogen fixation (Hayat et al., 2012a). Primarily, plant

growth promotion by PGPR occurs directly by either aiding in nutrients uptake

(nitrogen, phosphorus and other nutrient elements), by regulating the concentration

of growth regulating substances, or otherwise by minimizing the deleterious impact

of pathogenic microbes towards plant health hence serving as biocontrol agents

(Glick, 2012) (Table 2).

2.2.1.1 Phosphorus solubilization

Amongst the macronutrients, phosphorous (P) is considered as the main

growth-limiting nutrient element, and for its biological availability no significant

atmospheric source is present. The plant attributes related with phosphorus

availability are the process of N2-fixation in legumes, resistance towards plant

diseases and ensuring crop quality (Ezawa et al.,2002). It is present in the soil in

both organic and inorganic forms (Khan et al., 2009a) and inspite of its large supply

in the soil, the phosphorus available to the plants is very low. This low availability

is associated with the insoluble and adsorbed forms of P, dominatingthe soil P

26

reservoir. Plants can absorb P in only two soluble forms: monobasic (H2PO-4) and

dibasic ion diabasic (HPO2-4) (Bhattacharyya and Jha, 2012). P mechanism in the

27

Table 2:Beneficial effects on growth and yield of various crops by PGPR inoculation.

PGPR Crops Conditions Responses References

Pseudomonas sp. Mung bean Green house Increase peroxidase activity (82%) and reduce catalase activity(33%) in roots. chlorophyll a, chlorophyll b and total chlorophyll contents were significantly enhanced by 34, 48 and 39%, respectively

Sharma et al., 2003

Enterobacter aerogenes and Pseudomonas brenneri

Wheat Green house Improve root and shoot length, fresh and dry biomass per plant

Hayat et al., 2012b

Thiobacillus sp. and Rhizobium sp.

Groundnut Pot and field In pot experiment plant dry weight, nodulation and pod number was increased Under field condition nodule number, plant biomass and pod yield (18%) was augmented

Anandham et al., 2007

Pseudomonas spp. Groundnut Pot and field Increase in pod yield and soil N and P contents in pot experiment Under field conditions pod yield was increased (24%)

Dey et al., 2004

Burkholderia cenocepacia, Pseudomonas sp.

Rice Pot and field Enhanced shoot height (60%), biomass (33%), grain yield (up to 26%)

Jha et al., 2009

Serratia sp. and Rhizobium sp

Lettuce Greenhouse Alleviate the soil salinity impact and improve photosynthesis, mineral contents and plant growth

Han and Lee, 2005

Bacillus species, Paenibacillus polymyxa, Pseudomonas putida and Rhodobacter capsulatus

Sugar beet Green house Increase root weight by 46.7%. Leaf, root and sugar yield were enhanced by 20.8, 16.1, and 14.7% respectively

Çakmakçi et al., 2006

Rhizobium tropici, Phaseolus Green house Increase leghemoglobin concentration, Figueiredo et al., 2007

28

Bacillus sp., Paenibacillus sp.

vulgaris nitrogenase activity and N2 fixation efficiency.

Azotobacter sp. Cotton Field Enhance seed yield (21%), shoot length (5%) and soil microbial population (41 %)

Anjum et al., 2007

Pseudomonas fluorescensand Bradyrhizobiumsp.

Origanum majorana

Green house Shoot length, shoot and root dry weight was enhanced significantly. Oil yield was also increased

Banchio et al., 2008

Bacillus sp. Beta vulgarisand Hordeum vulgare

Field Improve yields (5.6-11.0%) Şahin et al., 2004

Bacillus licheniformis, Rhodobacter capsulatus, Paenibacillus polymyxa, Pseudomonas putida, and Bacillus sp.

Barley Green house Increase in root weight by (32.1%) and shoot weight by (54.2%). Improve uptake of N, Fe, Mn, and Zn. Soil NO3-N and P contents were increased

Çakmakçi et al., 2007

Bacillus firmis, Bacillus megaterium and Pseudomonas aeruginosa

Groundnut Pot and field Plant biomass was increased (26%) in pot experiment. Improved plant growth and pod yield (19%) was observed under field condition

Kishore et al., 2005

Bacillus sp., Pseudomonas sp. and Bradyrhizobium japonicum

Lupinas albus

Growth chamber

Increased nodulation and plant dry weight Lucas Garcia et al., 2004

Pseudomonas putida, Pseudomonas fluorescens and Bradyrhizobium sp.

Mung bean Axenic and in vitro

Increased nodulation and suppression of ethylene

Shaharoona et al., 2006

Azospirillum brasilense and Bradyrhizobium japonicum

Soybean and corn

Growth chamber

Increased seed germination and nodule formation

Cassàn et al., 2009

Bacillus subtilis and Bradyrhizobium

Soybean Green house and field

Augmentation of mass and nodule number, biomass, nitrogen contents and grain yield

Bai et al., 2003

29

japonicum Bacillus subtilis and Azospirillum brasilense

Tomato In vitro Regulate and increase root growth and development

Felici et al., 2008

Pseudomonas aurantiaca Wheat and maize

Field Improve microbial colonization in root and increase yield

Rosas et al., 2008

Pseudomonas sp. and Bacillus sp.

Wheat In vitro Increase root elongation (17.3%), root dry weight (13.5%), shoot elongation (37.7%) and shoot dry weight (36.3%)

Khalid et al., 2004

Bacillus sp. Raspberry Field Enhanced yield (33.9%), cluster number (28.7%), berries number (36.0%). Increase soil P contents

Orhan et al., 2006

Mung bean, mash bean and wheat

Axenic Biomass and grain yield in wheat (45%) and beans (50%) are improved significantly

Hayat et al., 2013

Kluyvera ascorbata Tomato, canola and mustard

Axenic Minimize the deleterious effects of nickel, zinc and lead by synthesizing siderophores

Burd et al., 2000

Bacillus spp. Tomato, bell pepper, watermelon, sugar beet, tobacco,Arabidopsis sp.

Green house Protection from leaf-spotting fungal and bacterial pathogens, systemic viruses, crown-rotting fungal pathogen, root-knot nematodes, and a stem-blight fungal pathogen in addition to damping-off, blue mold, and late blight diseases

Kloepper et al., 2004a

Pinus contorta

Axenic Stimulated growth and associative nitrogen fixation

Chanway and Holl, 1991

Pseudomonas putida Canola Axenic Enhanced root elongation and the plants were able to grow at 5 °C

Sun et al., 1995

Pseudomonas sp. Wheat In vitro Plant height, root and shoot biomass and number of tillers was increased

de Freitas and Germida, 1990

Mesorhizobium sp. and Pseudomonas aeruginosa

Chickpea P and Iron (Fe) uptake was enhanced. Minimize the deleterious effects of wilt and root rot. Increased nodulation (86%),

Verma et al., 2014

30

root dry weight (57%), shoot dry weight (45%), grain yield (32%), straw yield (41%), N (65%) and P (59%) uptake

31

soil is depicted by absorption-desorption (physicochemical) andimmobilization-

mineralization (biological) activities. The insoluble P fraction is bound on inorganic

minerals such as apatite or is present in organic forms like inositol phosphate,

phosphotriesters or phosphomonesters (Glick, 2012). Repeated applications of

phosphatic fertilizers in soil are performed to overcome this deficiency. Plants

absorb very less amount of applied fertilizer and remaining penetrates into the

stationary pools by precipitation reaction with Al+3 and Fe+3 ions in acidic and Ca+2

in calcareous soils (Hao et al., 2002). Soil bacteria capable of phosphate

solubilization, commonly known as phosphate solubilizing bacteria (PSB), perform

a crucial part in soil P dynamics and P accessibility for the roots, hence proved to

be a potential alternative of chemical fertilizers (Khan et al., 2006). Since 1950s

PSB are being applied as biofertilizers in agricultural soils(Sial et al., 2015), and

bacteria belonging to genus Azotobacter, Bacillus, Beijerinckia, Burkholderia,

Enterobacter, Erwinia, Flavobacterium, Microbacterium, Pseudomonas,

Rhizobium and Serratia are documented to be the most efficient PSB

(Bhattacharyya and Jha, 2012). Generally, these bacteria release low molecular

weight organic acids which disintegrate phosphatic minerals or chelate cationic P

ions i.e., PO4-3, thereby releasing P in the soil solum (Zaidi et al., 2009) (Figure 2).

There exists a synergetic association between plants and PSB, since soluble

phosphates are supplied by the bacteria and plants provide carbon sources (mostly

carbohydrates), utilized by bacteria for their metabolic processes (Pérez et al.,

2007). The beneficial impact of PSB inoculation used alone (Poonguzhali et al.,

2008; Chen et al., 2008; Ahemad and Khan, 2012) or in consortium with other

PGPR (Zaidi and Khan, 2005; Vikram and Hamzehzarghani, 2008) has reported an

32

Figure 2: Different compounds released by PSB accounted for P solubilization in the soil

33

increase in crop yield as a result of improved efficacy of BNF and by increasing the

accessibility of other nutrient elements by releasing important PGP substances

(Zaidi et al., 2009). Combined inoculation of PSB and arbuscular mycorrhiza

resulted in better P uptake resulting its release from insoluble soil pool and also

from rocks rocks (Goenadi et al., 2000; Cabello et al., 2005). Increase in plant yield

upto 70% is reported due to solubilization of insoluble soil P by PSB by Verma

(1993). Son et al. (2006) revealed significant increase in different yield and growth

components of soyabean like enhanced nodulation, biomass, nutrients uptake and

yield by the application of Pseudomonas sp. PSB improved the seedling height in

chick pea (Sharma et al., 2007), whereas co-inoculation of PSB with PGPR in corn

lower the utilization of phosphorous fertilizer to half, with no negative effect on

yield (Yazdani et al., 2009). In green gram combined application of

Bradyrhizobium, Glomus fasciculatum and Bacillus subtilis enhanced grain yield

by 25%(Zaidi and Khan, 2006). PGPR can interact beneficially in plant growth

promotion, along with improved N and P uptake (Jilani et al., 2007). Therefore, use

of PSB as biofertlizer has huge potential to utilize the soil fixed P and reserves of

phosphate rocks (Khanet al., 2009b).

2.2.2.2 Phytohormone production

PGPR may stimulate plant growth by the synthesis and secretion of plant

growth regulating substances (Hayat et al., 2010). The bacteria produce and export

plant hormones known as plant growth regulators (PGRs). PGRs are organic

exudates having positive impact on plant physiological processes at very minute

concentrations (Dobbelaere et al. 2003). PGRs are classified into five classes,

auxins, gibberellins, cytokinins, ethylene and abscisic acid (Zahir et al., 2004).

34

Among these phytohormones significant emphasis is given to auxin. Among

all naturally occurring auxins, indole-3-acetic acid (IAA) has been reported to be

synthesized by 80% of soil bacteria (Patten and Glick, 1996), and majority of the

literature considers IAA and auxin as synonyms (Spaepen et al., 2007). IAA is

known to effect plant cell division, cell elongation and differentiation, influences

seed germination, stimulates the xylem and root formation and development

(consequently enhancing plants accessibility to soil nutrients) , regulates vegetative

growth and offer resistance to stressed environment (Tsavkelova et al.,

2006).Considering the potential of IAA in plant growth, its synthesis by soil

bacteria could be considered as one of the approach to evaluate efficient plant

growth promoting strains (Khalid et al., 2004). This diversity in functions

performed by IAA is attributed to its complex biosynthetic, transport and signaling

pathways (Santner et al., 2009). The synthesis of IAA is altered by an amino acid

called tryptophan, recognized to be an important precursor for its biosynthesis

(Zaidi et al., 2009). Supplementation of bacterial culture media with tryptophan

resulted in increased production of IAA by most rhizobacteria (Spaepen and

Vanderleyden, 2011). IAA has been reported to synthesize by many Rhizobium sp.

(Ahemad and Khan, 2011, Ahemad and Khan, 2012). As IAA is responsible for cell

division and segregation and formation of xylem and phloem and these mechanisms

are involved in nodule development, therefore auxin level in legume plants is

mandatory for nodule formation (Glick, 2012). Environmental factors modulating

IAA synthesis in bacteria comprise of low pH, osmotic and matrix potential and

carbon regulation (Spaepen et al., 2007). Genetic factors controlling IAA

production include the location of auxin synthesis gene in bacteria (either on

plasmid or chromosome) and the mean of expression (induced or constitutive). This

35

location of auxin synthesis gene can affect the IAA production level, as plasmids

are present in multiple copies, hence resulting in increased IAA level in bacteria.

The first genus of bacteria attributed to be used as PGPR and release IAA to prompt

plant growth wasRhizobium (Mandal et al., 2007). Sridevi and Mallaiah (2007)

have reported the synthesis of IAA from all Rhizobium species isolated from

Sesbania sesban nodules. Rhizobium sp. isolated from Vigna mungo resulted in

increased IAA level to root nodules (Mandal et al., 2007), exhibiting that Rhizobia

could be used as PGPR for crop production as it can improve root system and

nutrients uptake (N, P and K) by synthesis of IAA. Other IAA producing bacteria

belong to genera Azospirillum, Aeromonas, Azotobacter, Bacillus, Burkholderia,

Enterobacter and Pseudomonas (Shoebitz et al., 2009).

The plant hormone ethylene is an efficient PGR, synthesized by almost all

bacterial species (Alonso and Ecker, 2001), and is active at very low concentration

(0.05 ppm) (Abeles et al., 1992). Ethylene can affect plant growth and senescence

(Miransari, 2014) by various means including fruit ripening, promotes root

initiation, stimulates seed germination, activates the synthesis of other plant growth

regulators and induces resistance to biotic and abiotic stresses. The ethylene

secreted as a result of stresses is called “stress ethylene”, and its synthesis is linked

to extreme changes in environment like high temperature and light, dryness,

prevalence of toxic metals, insect predation, high salt concentration etc (Wang et

al., 2013b). If ethylene higher concentration persists after germination, it inhibited

root elongation and symbiotic N2-fixation in legumes. Therefore, many PGPR may

prompt plant growth by reducing ethylene level in plants by synthesizing enzyme

1- aminocyclopropane-1-carboxylate (ACC) deaminase (ACC deaminase),

36

resulting in hydrolyses of ACC, which is the precursor of ethylene in plants (Sabir

et al., 2013), thus reducing the negative effects of high ethylene concentrations.

PGPR exhibiting ACC deaminase activity improved plant growth and yield,

reduced ethylene concentration and drought stress and induced salt tolerance, hence

and can effectively be used as biofertilizer (Shaharoona et al., 2006). The bacterial

strains illustrating ACC deaminase activity are known to belong to genera

Acinetobacter, Achromobacter, Agrobacterium, Alcaligenes, Azospirillum,

Bacillus, Burkholderia, Enterobacter, Pseudomonas, Ralstonia, Serratia and

Rhizobium etc. (Zahir et al., 2009; Kang et al., 2010). These bacteria consume ACC

resulting its transformation into 2-oxobutanoate and NH3 (Arshad et al., 2007). The

significant effects of ACC deaminase synthesizing rhizobacteria are root elongation

and shoot growth, increased nodule number and nutrients uptake and improved

colonization of mycorrhiza in many plants (Glick, 2012).

Many studies have revealed that almost all rhizobacteria have the potential

to synthesize cytokinins or gibberellins or both (Saharan and Nehra, 2011).

Cytokinins have been reported to be produced by bacteria belonging to genera

Azotobacter, Rhizobium, Pantoea, Rhodospirillum, Pseudomonas, Bacillus and

Paenibacillus. Cytokinins exhibit various effects when applied to the plants.

Primarily, they activate protein synthesis and as a result they enhance chloroplast

maturation and delay the senescence. The beneficial effect of cytokinins can be

observed in tissue culture along with auxin, where they activate cell division and

regulate morphogenesis. When added to shoot culture medium, they alleviated

apical dominance and resulted in release of lateral buds. The mode of action of

cytokinins at molecular level is not well-known, even though they perform dynamic

37

functions in plant growth and development. In many cases, they stimulate RNA

synthesis, thus activating protein and enzymes activites (Brenner et al., 2012).

More than 100 different types of gibberellins are now known. They are tetracyclic

diterpenoid acids, all possessing gibbane ring arrangement and they may be

dicarboxylic (C20) or monocarboxylic (C19). Gibberellins perform many important

plant functions including stem elongation, initiation of seed enzymes like α-

amylase and protease thus facilitating endosperm metabolism, promotion of seed

germination and fruit development (George et al., 2008). Not only fungi and higher

plants are responsible for the synthesis of gibberellins but bacteria also synthesize

and sectrete this hormone (MacMillan, 2002). There is no recognized function of

gibberellins in bacteria; however they behave as secondary metabolites and act as

signaling agent for the host plant. The occurrence of GA1, GA4, GA9 and GA20 is

revealed in Rhizobium meliloti by Atzorn et al. (1988), GA1, GA3, GA9, GA19 and

GA20 have been identified in Azospirillum sp. under gnotobiotic studies (Piccoli et

al., 1997). Besides Azospirillum and Rhizobium sp., gibberellins production is also

documented by bacteria belonging to genera Bacillus, Acetobacter and

Herbaspirillum (Jha and Saraf, 2012).

Another naturally existing regulator is abscisic acid (ABA), composed of

C15 ring structure. Its naturally existing type is S-(+)-ABA. It is synthesized from

different carotenoids and xanthophylls, resulting in xanthoxin that is transformed

into ABA aldehyde then finally to ABA (Kepka et al., 2011). ABA is produced in

stressed conditions like dehydration of plant tissues, extreme temperatures, salt

concentration and high water table (Tuteja, 2007), leading to the rising concept in

research of rhizobacteria, resulting in their classification as “Plant Stress Homeo-

38

regulating Rhizobacteria (PSHR)” (Cassán et al., 2005). In the beginning, ABA

was studied to effect bud dormancy and abscission, therefore generally it is

considered as plant growth inhibitor; however it performs many functions in plants

like govern stomatal opening, stimulate storage protein synthesis and regulate water

and ion uptake by the roots (Schachtman and Goodger, 2008). ABA synthesis was

reported by bacterial strain Azospirillumbrasilense(Perrig et al., 2007).

2.2.2.3 Nitrogen fixation

Among the macronutrients required for crop growth, nitrogen (N) is the

most essential for acquiring optimum crop yield. The atmospheric nitrogen (78%)

can only be utilized by the plants only when it is converted into ammonia or nitrate

form. This is achieved by the process of biological nitrogen fixation (BNF),

performed by bacteria possessing nitrogenase enzyme system converting nitrogen

into ammonia (Swain and Abhijita, 2013). BNF occurs by the action of nitrogen

fixing microorganismsrepresenting an environment friendly substitute to chemical

fertilizers (Carvalho et al., 2014). Nitrogen fixing microorganisms are mainly

classified as (i) symbiotic N2-fixing bacteria of the family Rhizobiaceae which exist

in symbiotic association with legume plants (Ahemad and Khan, 2012) and (ii) free

living, associative and endophytic nitrogen fixers including cyanobacteria

(Anabaena), Azospirillum, Azotobacter etc. (Bhattacharyya and Jha, 2012);

although non-symbiotic nitrogen fixers fixed very small amount of nitrogen (Glick,

2012). The use of PGPR as bio-fertilizer along with nitrogen fixing bacteria not

only reduces the application of chemical fertilizer but also lowers the input cost,

thus prove to be potential alternative to chemical fertilizers in addition to preserve

the environment (Stefan et al., 2008).

39

2.2.2.3.1 Symbiotic nitrogen fixing bacteria

The nitrogen fixing bacteria belong to genus Rhizobium of family

Rhizobiaceae infect nodules and exist in symbiotic association in roots of legumes.

Numerous complex processes are responsible for this symbiosis to develop

(Udvardiand Poole, 2013), as a result of which nodules are formed. The enzyme

nitogenase complex is responsible for the process of biological nitrogen fixation

(Weisany et al., 2013). It consist of (i) dinitrogenase reductase composed of iron

protein complex, provides highly reduced electrons and (ii) dinitrogenase

possessing a metal as cofactorand uses the electrons provided by dinitrogenase

reductase to reduce N2 to NH3+. Three different N-fixing systems have been

recognized based on the metal cofactor i.e., (i) Mo-nitrogenase, (ii) Fe-nitrogenase

and (iii) V-nitrogenase; Mo-nitrogenase is present in all diazotrophs hence playing

a major contribution in BNF (Ahemad and Kibret, 2014). Nitrogen fixing genes

(nifH genes) are present in symbiotic as well as free living bacteria. Nitrogenage

(nifH) genes are responsible for activation of Fe protein, synthesis of Fe-Mo

cofactor, and synthesis and functioning of the enzyme. These genes exist in a

cluster of 20-24 kb, encoding twenty different proteins (Glick, 2012).

Even though rhizobia effectively nodulate legumes, some species can exist

in symbiosis with non-legume plants like Parasponia (Wang et al., 2012). The

process of colonization of non-legumes by rhizobia is different than Rhizobium-

legume symbiosis, as it started from rhizosphere into the epidermis, endodermis

and cortex, but the primary place of colonization is intercellular region of the roots.

The non-specific association of rhizobia with roots of non-legume plants identified

the diversity of this genus to behave as PGPR. More often endophytes are more

40

competitive root colonizer and are capable of competing the surrounding bacteria

(Gaieroet al., 2013). Field studies conducting on rhizobia-legume symbiosis in

India portrayed that half dose of nitrogen fertilizer may possibly be minimized by

rhiozbial inoculation resulting in significant yield enhancement (Tilak, 1993).

Rhizobia can enhance P uptake by the plants by solubilizing inorganic and organic

phosphates in the soil (Alikhani et al., 2006) and co-inoculation of Rhizobium sp.

with PSB resulted in improved symbiosis and increased grain yield of mung bean

and raise the competitive potential and symbiotic efficiency of Rhizobium sp. in

lentil in field experiments (Kumar and Chandra, 2008). Another PGPR

Azorhizobium caulinodans and Azospirillum brasilense enters the rice roots by

crack entry and move to cortical root cells, the bacterial entry and movement into

the roots is triggered by flavonoids molecules released by rhizobium (Brencic and

Winans, 2005). Some soil indigenous rhizobia can occupy the emerging roots of

wheat,oilseed rape, rice and maize crops (Cocking et al., 1990, 1994). Rhizobia

belonging to genera Rhizobium and Bradyrhizobium synthesize and release plant

growth promoting molecules like abscicic acids, auxins, cytokinins, riboflavin and

lipochitooligosaccharides which resulted in improved plant growth and increased

grain yield (Hayat etal., 2008a, b; Hayat et al., 2010). Rhizobia are also capable to

release siderophore (Brearet al., 2013), solubilze insoluble phosphates and protect

the plant from soil pathogens (Ahemad and Kibret, 2014).

2.2.2.3.2 Non-symbiotic nitrogen fixing bacteria

Non-symbiotic N2-fixation proved to be agronomically important, although

the carbon and ATP availability for this process to occur is the major limitation.

This can be overcome by the movement of bacteria inside the plant rhizosphere or

41

rhizoplane. The main non-symbiotic N2-fixing bacteria consist of bacteria

belonging to genus Azoarcus, Herbaspirillium, Azotobacter, Achromobacter,

Acetobacter, Azospirillum, Azomonas, Bacillus, Beijerinckia, Clostridium,

Corynebacterium, Derxia, Enterobacter, Klebsiella, Pseudomonas, Rhodospirillum,

Rhodopseudomonas and Xanthobacter (Santi et al., 2013). Azospirillum are aerobic

nitrogen fixing bacteria living in plant roots vicinity. Inoculation of Azospirillum in

wheat under greenhouse and field experiments has resulted in increased yield

(Ganguly et al., 1999) This increase in yield is ascribed to improved root

development resulting in augmentation of water and nutrients uptake and biological

nitrogen fixation (Ghany et al., 2014). The synthesis of phytohormones by

Azospirillum manipulate the plant root reparation and proliferation rate, metabolism

and finally nutrients uptake. Various species of Azospirillum including Azospirillum

lipoferum, Azospirillum brasilense and Azospirillum amazonese have been reported

to isolate from paddy and sugarcane (Sivasakthivelan and Saranraj, 2013).

Azospirillum lipoferum reported to increase rice yield upto 6.7 g plant-1 in

greenhouse studies (Mirza et al., 2000) and wheat grain yield was increased by

30% as a result of Azospirillum brasilense inoculation Okon and Labandera-

Gonzalez, 1994).

Another diazotrophic acid-tolerant endophyte is Acetobacter and

Acetobacter-sugarcane system is an efficient model for BNF dominated by nifH

gene (Ohyamaet al., 2014). An endophytic Gram-negative diazotrophic bacteria

Azoarcus sp. was isolated from Kallar grass grown in Pakistani soils of saline-sodic

nature (Reinhold-Hurek et al., 1993). The genus Burkholderia consist of 90

validated species, with most species possessing the potential of nitrogen fixation

42

(Vandamme et al., 2002).Burkholderia vietnamiensis when co-inoculated with rice

crop economize 25-30 kg ha-1 N (Tran Vân et al., 2000). The family

Enterobacteriaceae includes diazotrophs, and contains PGR synthesizing genera

including Enterobacter, Pseudomonas, Klebsilla and Citrobacter mostly isolated

from rice rhizosphere (Kennedy et al., 2004). One of the common bacterial genus

in agricultural soils is Pseudomonas and possess many attributes which render them

as good PGPR. Among them the effective species include Fluorescent

pseudomonas (FLPs) which maintain soil health and metabolically active (Lata et

al., 2002). Chickpea yield and growth is enhanced by FLPs in combination in

Rhizobium sp. (Rokhzadiet al., 2008), and FLPs isolated from sugarcane

rhizosphere significantly increase plant biomass (Mehnaz et al., 2009). Plant roots

of potato, radish and sugar beet are effectively colonized by Pseudomonas sp.

resulting in significant increase in yield upto 144% under field conditions

(Kloepper et al., 2004b).

The most abundant bacterial genus in the rhizosphere is Bacillus and their

plant growth promoting activities are recognized for many years (Gutiérrez Mañero

et al., 2003). Several PGR substances are secreted by these strains in the soil

(Charest et al., 2005), resulting in improved nutrients uptake by the plants. Bacillus

subtilis is present naturally in the rhizosphere of plant roots and effectively sustain

interaction with plants to improve their growth. García et al. (2004) while

inoculating Bacillus licheniformis on tomato and pepper observed significant

colonization on the roots and rendered it a potential biofertilizer. The inoculation of

Bacillus megaterium (PSB) and Bacillus mucilaginosus in nutrients limiting soil

43

confirmed that they improve mineral availability, recommending their

implementation as biofertilizers (Han et al., 2006).

2.3 APPLICATION OF PGPR AS BIOFERTILIZER

As soil is an indeterminate natural environment and the desired results are

not always achieved, therefore the impact of PGPR application is different under

lab, green house and field conditions. The profound variations in climate affected

the efficiency of PGPR, but the undesirable field conditions are rendered as normal

characteristics of agriculture (Zaidi et al., 2009). Plant growth promoting attributes

function in synergistic phenomenon, and it is suggested that numerous mechanisms

like nitrogen fixation, P-solubilization, phytohormone production, siderophore

synthesis, ACC deaminase and anti-fungal activity etc. are collectively accounted

for the enhanced plant yield and growth promotion (Ahemad and Kibret, 2014).

Considerable increase in plant yields has been observed by the use of PGPR in both

natural and controlled conditions. The broader use of PGPR may reduce the

reliance on chemical fertilizers and pesticides in sustainable agriculture. Moreover,

this technology is easily approachable to the farmers especially in developing

countries (Gamalero et al., 2009).

One of the major challenges to encounter is the use of PGPR for their

commercial application. This encompasses the procedures and practices to maintain

the quality, reliability and productivity of biofertilizer. In this regard, B. subtilis has

been in constant use for formulation of commercial biofertilizer (Malusá et al.,

2012). In the development of potential PGPR biofertilizer, the main focus of the

researchers is to assure their endurance and activity under field conditions. Many

44

limitations are still there regarding registration and marketing of biofertilizers

(Malusá and Vassilev, 2014). In Pakistan, National Institute for Biotechnology and

Genetic Engineering (NIBGE) has registered and commercialized the bacterial

biofertilzer named as “Biopower” to be used for different leguminous and non-

leguminous crops like rice, wheat, chickpea, soybean, maize, cotton etc. National

Agricultural Research Centre (NARC) has commercialized biofertilizer under the

tag of “Biozote”; Biozote-N for leguminous crops, Biozote-P for all field crops and

Biozote-Max for non-leguminous crops. Different bacterial genera are being

considered as PGPR, but more focus is on Pseudomonas and Bacillus. Many

potential species of Bacillus are temperature tolerant; therefore they can prolong

more in commercial form, hence are extensively used in biofertilizers. At present,

much research is requisite for extensive commercialization of PGPR biofertilizers,

mainly because of their poor response under different field conditions (Malusá and

Vassilev, 2014)and among different plant cultivars (Remans et al., 2008).

Therefore there is a dire need for indepth study on colonization and functioning of

bacteria under variant soil and climatic conditions to ensure their potential use as

biofertilizers.

45

Chapter 3

MATERIALS AND METHODS

The Pothwar plateau(575 m average altitude; 32° 10' to 34° 9' N; 71º 10'to

73º 55'E) comprises of districts Jhelum, Chakwal, Rawalpindi (including ICT) and

Attock of northern Pakistan (Nizami et al., 2004). Groundnut is cash crop of

resource poor farmers of district Attock and Chakwal yielding 85% of country

groundnut. The average annual rainfall in these districts varies from 300 to 510

mm. The soils are calcareous in nature having been developed from loess and sand

stone parent material which varies from moderately coarse to moderately fine in

texture, thus suitable for pod development. The soil fertility is quite low. The

present study was carried out to isolate native strains of rhizobacteria from nodules

and rhizospheric soil of groundnut grown in Chakwal and Attock districts of

Pothwar. Isolated bacterial strains were screened based on their molecular,

biochemical and PGP traits to find most potential PGPR. Novel discovered strain

was also characterized for its physiological traits and international collaboration

was developed for chemotaxonomic analysis to fulfill the minimal standards for

description of new taxa.Green house and field studies were carried out to

investigate the co-inoculation effect of most efficient PGPRs and Rhizobium for

improving growth, yield and N2-fixation of groundnut. A detailed description of

methodology adopted in these studies is given in thischapter.

3.1 SURVEY FOR SAMPLE COLLECTION

Extensive farmer’s field survey was carried out for the collection

ofrhizospheric soil and nodules of groundnut from Attock and Chakwal districts in

2011. Rhizospheric soil and groundnut roots (to a depth of 15 cm) were collected

43

46

from farmers’ fields of Haji Shah, Ghazi Barotha, Sarwala, Sanjwal, Kamra, Dhok

Banaras, Groundnut Research Station, Attock, Fath-e-Jhang, Khunda, Pindi Gheb

of district Attock; University Research Farm, Koont, Dhudial, Barani Agricultural

Research Institute of district Chakwal. The sampling was completed during the pod

development stage and intact plants were excavated along with their roots. Roots

were gently removed and washed carefully so as to remove the adhered soil

particles completely. The washed roots were placed in properly labeled plastic

bags, stored in ice box and transported to laboratory for bacterial isolation.

3.2 ISOLATION AND PURIFICATION OF RHIZOBACTERIA FROM

SOIL AND NODULES

3.2.1 Isolation and Purification of Rhizobacteria from Rhizospheric Soil

Around 80 bacterial strains were isolated from rhizospheric soil by using

dilution plate technique where Phosphate Buffer Saline (PBS, 1X) was used as

saline solution. The bacterial isolates were allowed to grow on Tryptic Soya Agar

(TSA; Difco) medium (Table 3). These plates were incubated at 28 °C for 2-3 days.

After bacterial growth individual colonies were picked and streaked on plates

containing TSA media for purification under sterilized conditions in laminar air

flow cabinet. Single colonies were repeatedly re-streaked till the purified cultures

were obtained (Figure 3) (Hayat et al., 2013) and they were designated according to

their source and site.

3.2.2 Isolation and Purification of Bacteria from Groundnut Nodules

For isolation from groundnut nodules, healthy and unbroken nodules were

47

separated from the roots (from each nodule side, 0.5 cm root was cut). The surface

sterilization of nodules was performed by dipping in 95% ethanol for 5-10 seconds,

washed with sterilized distilled water for 3-4 times then immersed in 3% hydrogen

peroxide for 3-4 minutes. Nodules were crushed with blunt tipped sterilized forceps

on sterile petri plates and small amount of the nodule suspension was streaked on

petri plates having yeast mannitol agar (YMA) (Table 3). These plates were put in

incubator set at 28 °C for 2-3 days. Individual bacterial colonies were picked with

sterilized loop and were streaked on the media plate for purification (Carter and

Gregorich, 2007).

3.3 MAINTAINCE OF BACTERIAL CULTURE

Most of the axenic bacterial cultures obtained from the rhizospheric soil and

root nodules were maintained in petri plates containing respective media and stored

at4 °C. The bacterial strains were maintained in 35% w/v glycerol at -80 °C for

long lasting storage. The strain proposed as novel species in this study was

deposited in internationally recognized culture collection centers: Culture

Collection of China Agriculture University (CCBAU, China) and Laboratorium

voor Microbiologie, Universiteit Gent (LMG, Belgium).

3.4 PLANT GROWTH PROMOTING CHARACTERIZATION

PGPRs isolated from rhizospheric soil and nodules were characterized for

different plant growth promoting traits.

3.4.1 Phosphate solubilization

Nautiyal medium (Table 4) was prepared in petri plates to screen all the

48

Figure 3: Schematic diagram for the isolation of bacteria from groundnut

rhizospheric soil.

Incubation at 28°C

Series of re-isolation steps

Pure bacterial isolates

Incubation at 28°C

Isolation

49

Table 3:Composition of media used for culturing rhizospheric and nodule bacteria

Tryptic Soy Agar (TSA) (Difco) Quantity g L-1 pH 7-7.5

Tryptone (Pancreatic Digest of Casein) 15.0g Soytone (Papaic Digest of Soybean Meal) 5.0g NaCl 5g Tryptone (Pancreatic Digest of Casein) 15.0g

Yeast Mannitol Agar (YMA) Quantity g L-1 pH 7

Yeast Extract 3g

Mannitol 10g

KH2PO4 0.25g

K2HPO4 0.25g

NaCl 0.1g

MgSO4.7H2O 0.1g

Agar 15g

50

isolates on the basis of their phosphate solubilizing ability (Nautiyal, 1999).

Quantitative evaluation of bacterial strains to solubilize tricalcium

phosphate was performed following the procedure described by Kuo (1996). The

tested isolates were inoculated in 100 mL Nautiyal broth containing 0.5%

tricalcium phosphate and incubated for 7 days at 28 °C. The pH of the broth was

recorded before inoculation and after full growth. The centrifugation of fully grown

bacterial cultures was performed at 3000 rpm for 30 min. The available P in the

supernatent was determined following the procedure of Nautiyal (1999). To 2 mL

of bacterial supernanent, 2 mL reagent A (0.1 M ascorbic acid 8.8 g +

trichloroacetic acid (C2HCl3O2 0.5M) 40.9 g), 400µL reagent B (0.01M ammonium

molybdate 6.2g) and 1000 µL reagent C (sodium citrate (Na3C6H5O7.2H2O 0.1 M)

29.4g + sodium arsenite (NaAsO2 0.2M) 26g + (5%) glacial acetic acid (99.9%) 50

mL) was added. Standards were prepared by KH2PO4 and absorption was taken at

spectrophotometer at 700 nm.

3.4.2 Synthesis of Indole-3-Acetic Acid (IAA)

The ability of the isolated strains to synthesize IAA was estimated as

reported by Brick et al. (1991). Bacterial strains were cultured in test tubes

containing 5 mL Luriya Broth (LB) with and without the supplement of tryptophan.

The cultures were set to shake at 28 °C for 48-72 h. The bacterial cultures were

centrifuged at 3000 rpm for 30 min. Two drops of orthophosphoric acid was added

in 2 mL of the supernatant along with the addition of 4ml Salkowski reagent

(50mL, 35% of perchloric acid, 1mL 0.5M FeCl3 solution). The appearance of

pinkish color in thesolution indicated IAA production ability of the isolates.

51

Table 4: Composition of Nautiyal media.

Nautiyal media Quantity g L-1 pH 7

Glucose 10 (NH4)2SO4 0.5 MgSO4.7H2O 0.1 KCl 0.2

NaCl 0.2

FeSO4.7H2O 0.002

MnSO4. H2O 0.002

Ca3(PO4)5 0.5

52

The optical density was measured at 530 nm by making use of spectrophotometer

(Spectronic 20D). Standard graph of IAA (Hi-media) within 10-100 µg mL-1 gave

the IAA concentration produced by the cultures.

3.4.3 Amplification of nifHGene

The ability of the bacterial isolates for nitrogen fixing nifH gene was

determined through PCR (Figure 4) using reverse and forward nifH gene primers

(Table 5) (Poly et al., 2000).

3.5 BIOCHEMICAL AND PHENOTYPIC CHARACTERIZATION

Gram staining, catalase and oxidase synthesizing ability of the bacterial

isolates were determined by using (bioMérieux) kits according to the procedure

described by Cowan and Steel (1996). The potential of the bacteria to use a

particular substrate was assessed using the API ZYM, API 20E (bioMérieux) and

Biolog GN2 Microplate (Biolog) followed by manufacturer’s recommendations.

Individual colonies were subcultured on media plates to achieve full growth at 28

°C and were read at 6, 24 and 48 h with a computer controlled MicroPlate reader.

The procedures were carried out in the Laminar Flow Hood to ensued sterilized

environment.

The temperature range was calculated by incubating cultures in respective

medium at different temperatures (4, 10, 15, 20, 28, 37, 50, 60 °C). The pH range

was determined using tryptone yeast extract (TY) broth media with pH between 2.0

and 12.0 (at 1 pH unit increase). 1 N HCl or 1 N Na2CO3 was used to adjust the pH

and were confirmed after autoclaving. The salinity range for growth tolerance was

53

Initial Denaturation 96 °C, 5min

Denaturation 94 °C, 10sec

Annealing 54 °C, 5sec x 35 cycles

Elongation 72 °C, 4min

Storage 4 °C

Figure 4:PCR cycling conditions for nifH gene amplification.

54

studied in respective medium containing 0–5 % (w/v) NaCl at 1% increment.

Resistance of isolated strains to antibiotics was determined at 5, 50, 100 and

300 µg mL-1 for seven antibiotics i.e., tetracycline hydrochloride, neomycin sulfate,

chloramphenicol, ampicilin, kanamycin sulfate, streptomycin sulfate and

erythromycin. The plates were placed in incubator at 28 °C for 48-72 h and zone of

inhibition or clearing surrounding the discs was recorded as antibiotic sensitivity.

3.6 IDENTIFICATION USING MOLECULAR TECHNIQUES

All the isolates from rhizospheric soil were identified using molecular tagging

of 16S rRNA gene sequencing. Nodules bacteria were identified using 16S rRNA

biological marker as well housekeeping genes followed by bioinformatics and

obtaining accession number of all isolates from GenBanks.

3.6.1 Preparation of DNA Template

DNA template was prepared by picking individual colonies with the help of

sterilize toothpick and dissolve in 0.2 mL ependorf tube containing 1X Tris EDTA

(Ethylenediamine tetra acetic acid), boiled at 95°C to obtain DNA template

(Ahmed et al., 2007).

3.6.2 PCR amplification for 16S rRNA and housekeeping genes

The isolated strains were identified using 16S rRNA and protein-coding

housekeeping genes analysis. PCR amplification of DNA for 16S rRNA gene

sequencing was done followed by the procedure of Katsivela et al. (1999)

employing universal forward and reverse primers. The PCR amplification and

55

sequencing of partial atpD(ATP synthase subunit beta, 510 bp), recA (recombinase

A protein, 530 bp) and glnII (glutamine synthetase II, 600 bp) genes was performed

as stated by Gaunt et al. (2001) and Turner and Young (2000). The primers used for

PCR amplification of partial 16S rRNA, atpD,recA and glnII genes are given in

Table 5. The PCR mixture was prepared as given in Table 6, and the procedure of

PCR amplification of partial 16S rRNA, atpD,recA and glnII genes was performed

as shown in Figure 5 and 6.

Amplified PCR products for each gene were separated on 1% agarose gel in

0.5 X TE (Tris-EDTA) buffer containing 2 µL ethidium bromide (20 mg mL-1). The

biological marker λ Hind-III was used as ladder size marker. The gel was viewed

under UV light and photographed using gel documentation system (Ahmed et al.,

2007).

3.6.3 Sequence Analysis

16S rRNA gene sequencing was performed from MACROGEN, (Korea)

and BGI technology corporation, China; and for housekeeping gene analysis,

amplified DNA of nodule bacteria were sent to BGI technology corporation, China.

The sequences derived from each sequencing primers were checked, edited

and aligned by making use of BioEdit ver.7.0.5.3 (Hall, 1999). The almost

complete sequences of 16S rRNA, atpD, recA and glnII genes were submitted in

DNA Data Bank of Japan (DDBJ) using the Sakura Nucleotide Data Submission

System to obtain accession number of each strain.

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Table 5:Primers used for PCR amplification and sequencing.

Gene Nucleotide sequence

nifH PolF 5́TGCGAYCCSAARGCBGACTC3́ PolRb 5ʹATSGCCATCATYTCRCCGGA3́ 16S rRNA 1510R 5́-GGCTACCTTGTTACGA-3́ 9F 5́-GAGTTTGATCCTGGCTCAG-3́ (P6) 5ʹ-TACGGCTACCTTGTTACGACTTCACCCC-3ʹ (P1) 5ʹ-AGAGTTTGATCCTGGCTCAGAACGAACGCT-

3ʹ atpD atpD255F 5ʹ-AACGTC GCTSGGCCGCATCMTS-3ʹ atpD782R 5ʹ-GCCGACACTTCMGAACCNGCCTG-3ʹ recA recA-F6 5́-CGKCTSGTAGAGGAYAAATCGGTGGA-3́ recA-555 5ʹ-CGRATCTGGTTGATGAAGATCACCAT-3ʹ glnII glnII12F 5́-YAAGCTCGAGTACAT YTGGCT-3́ glnII689R 5ʹ-TGCATGCCSGAGCCGTTCCA-3ʹ

57

Table: 6: The composition of PCR mixture.

Composition µL DNA (template) 2 Takara Ex Taq 25 Forward primer 1 Reverse primer 1 DNA free water 21

58

Initial Denaturation 94 oC, 2min

Denaturation 94 oC, 1min

Annealing 55 oC, 1min x 30 cycles

Elongation 72 oC, 1min

Final Exrension 72 oC, 5min






Figure 5: PCR cycling conditions for 16S rRNA and atpD gene amplification.

59











Figure6: PCR cycling conditions for recAandglnII gene amplification.

60

3.6.4 Phylogenetic Analysis (Bioinformatics)

The sequence results obtained were blasted through EzBiocloud and

sequence of all the related species was retrieved to get the exact nomenclature of

the isolates. The sequences obtained were aligned along with the sequences of

closely related species obtained from GenBank by with the help of ClustalW

program in the MEGA 5.2 software (Tamura et al.,2007). Neighbor-joining un-

rooted phylogenetic trees were constructed by analyzing the aligned sequences by

means of MEGA 5.2 software and evolutionary distances were calculated using the

Kimura 2 parameter model (Nei and Kumar, 2000).

3.7 EVALUATE EFFECTIVENESS OF CO-INOCULATION OF PGPR

AND Rhizobium UNDER GREEN HOUSE AND FIELD

A pot experiment was conducted in greenhouse of Plant Breeding and

Genetics department in PMAS-Arid Agriculture University, Rawalpindi to

investigate the PGPR selected on the basis of their plant growth promoting traits on

growth, yield and N2-fixation of groundnut. Ten kg sterilized soil per pot was used

and the characteristics of the experimental soil are given in Table 7. The groundnut

seeds were surface sterilized with 3% H2O2, and treated with inoculums of seven

selected PGPRs along with Rhzobium isolated from groundnut (consortium). The

bacterial strains were grown in TSA medium plates in incubator for 48 hours. The

inoculum for treating seeds was prepared by suspending fully grown culture from

media plates into the liquid tryptic soy broth in incubator shaker. The suspended

cells were grown to attain about 109 cells mL-13(CFU) based on optical density OD

= 0.08. The inoculum was applied to sterile seeds before sowing and seeds were

immersed for 4-5 hours in the innoculm. Four seeds were grown in each pot and

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Table 7: Physicochemical characteristics of AAUR and Koont Site.

Characteristics AAUR Site Koont Site

Texture Silty clay loam Sandy clay loam

Sand (%) 19 56.0

Silt (%) 55 22.8

Clay (%) 26 21.2

EC (dS m-1) 0.31 0.53

Soil pH 7.7 7.87

Bulk Density (Mg m-3) 1.40 1.45

Total Organic Carbon (g 100g-1) 0.60 0.59

62

two seedlings in each pot were maintained after germination. Uninoculated pot was

considered as control. The experiment was carried out in completely randomized

design (CRD) with three replications. The parameters like nodule number, biomass

yield, pod yield, total shoot N% and total N2-fixed were estimated to confirm the

effect of inoculated PGPRs and Rhizobium on growth and N2-fixation of groundnut.

Field experiment was also conducted in summer 2013 in University Research

Farm Koont, at Chakwal road in summer 2013. Three most potential PGPRs were

selected on the basis of pot experiment along with Rhizobium, to investigate their

beneficial effect on groundnut yield and N2-fixation. The bacterial strains ratio in

consortium was 1:1:1. Six treatments were designed to check the integrated effect

of co-inoculation of PGPRs/Rhizobium along with full (NP @ 20 and 80 kg ha-1)

and half recommended dose (NP @ 10 and 40 kg ha-1) of chemical fertilizers. The

study was designed in randomized complete block design (RCBD) with three

replications, and plot size of 12 m2. The soil of experimental site was sandy clay

loam (Table 7). Groundnut variety BARI 2011 was sown with the seed rate of 35

kg acre-1. Bacterial inoculum was applied to the sterilized seeds before sowing.

The inoculum was prepared by suspending fully grown culture from media plates

into tryptic soy broth. The suspended cells were grown to attain about 109 cells mL-

13 (CFU).

For plant density, nodule number, total biomass and pod yield, crop was

harvested from 1 m2 quadrate randomly selected in each plot. The plant samples

were oven dried, weighed and total biomass was determined. For pod yield plant

samples were threshed manually and then total yield was recorded and converted to

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t ha-1.

3.8 PLANT ANALYSIS FOR TOTAL NITROGEN

Plant samples were dried in oven at 65oC for 48 hrs and grinded by

usingWiley Mill. Samples were stored in plastic containers for the determination of

N contents. For N2-fixation by natural15N abundance, grinded plant samples

ofgroundnut and reference weed plant growing in the same field were sent to

StableIsotope Unit, University of Waikato, Hamilton, New Zealand for15N analysis.

For total nitrogen, all digestion tubes were taken containing 0.2 g of ground

plant material and 4.4 mL of digestion mixture (selenium powder, lithium sulphate

and H2O2). The whole mixture was digested for two hrs at 360 °C. When the

solution became colorless 50 mL of water was added in it and mixed well. On

cooling, the volume was raised to 100 mL and mixing was done. The solution was

allowed to settle and clearer solution was used for analysis of total nitrogen

calorimetrically (Anderson and Ingram, 1993). For colorimetric determination of

total nitrogen (%), from each standard and sample 0.1 mL was taken and 5 mL of

reagent (sodium salicylate, sodium citrateand sodium tartarate and sodium

nitroprusside) was supplemented to them. The mixture was mixed well. After 15

min, in every test tube, another reagent (NaOH, water and sodium hypochlorite)

was incorporated and stand for an hour. The sample’s absorbance was measured

after complete color development by spectrophotometer adjusted at 665 nm. Plant

TN was assessed by using the equation given below:

TN % = C/W × 0.01

Wherein C is the corrected concentration (µg mL-1) and W is weight of sample (g).

64

3.9 ASSESSMENT OF N2-FIXATION BY NATURAL 15N ABUNDANCE

The %Pfix (proportion of N derived from air) was estimated using the

following equation.

%Pfix = 100 x 15N (soil N) - 15N (legume N)

(15N (soil N) –B)

Where:

15N (soil N) is attained from a non-N2 fixing reference plant (wheat) grown in the

same soil as the legume (adjusant plot, in same season).

B is the 15N of the same N2 fixing plants when grown with N2 as the only source

of N.

Legume N (kg ha-1) = legume total biomass (kg ha-1) x plant N%.

Total N fixed (kg ha-1) = Legume N (kg ha-1) x % Pfix x 1.5*

*1.5 factor is representing an estimate of contribution by below ground N (Peoples

et al., 1989).

3.10 SOIL ANALYSES

For field experiment, soil of experimental site (Koont) was analyzed for soil

texture (Gee and Bauder, 1986), pH (Thomas, 1996), ECe (Rhoades, 1996) total

organic carbon (Nelson and Sommers, 1996), Olsen P (Kuo, 1996) and nitrate-N

(Mulvaney, 1996) before sowing and after harvesting of plants.

3.11 VALIDATION OF NOVEL BACTERIAL SPECIES

After BLAST search in EzBiocloud, the nodule bacteria identified as

Rhizobium spp. (BN-19T), exhibited < 97% similarity with its closely related

identified strains was considered as potential novel candidate and validated by

65

adopting the proposed minimal standards for describing new taxa (Logan et al.,

2009). For this purpose, international collaboration (China Agricultural University,

Beijing, China through IRSIP HEC, Pakistan) was developed for chemotaxonomic

analysis along with five reference strains to fulfill the minimal standards for

description of new taxa. The reference strains were obtained from international

GenBank deposits.

3.11.1 Morphology and Phenotypic Characterization

Colony morphology was revealed by growing novel type strain on YMA for

3 days at 28 °C. Cell shape and size was visualized by scanning electron

microscope. For this purpose, cells were grown in TY medium at 28 °C for two

days, mixed in a solution of 1% glutaraldehyde in 0.01M phosphate buffer (pH 7.2)

for 2 h in room temperature. Cells were dispersed on the slide and air-dried. Water

from the cells was eliminated by dissolving the samples in acetone series (30, 50,

70, 90, 95 and 100) and 100% 2-methyl-2-propanol solution. After freeze-drying,

samples were coated with platinum under a vacuum, and cell morphology was

studied by using Hitachi S3400N scanning electron microscope.

Motility was determined by observing growth of cells in test tubes

containing semisolid YMA medium with 0.5 % agar concentration at 28 °C for 72 h

(Cowan and Steel, 1996). Gram staining was done following the manufacturer’s

instructions (bioMérieux, France). The generation time was calculated by culturing

bacteria in 5 mL TY media for 2-3 days. After sufficient growth, bacterial culture

was transferred to 100 mL TY media and optical density was recorded at 600 nm

after every hour to obtain exponential growth curve (Gao et al., 1994). For carbon-

66

source utilization, temperature, pH range determination, salinity tolerance and

resistance to antibiotics, the procedure described in section 3.5 was followed.

3.11.2 DNA Template for Amplification of 16S rRNA and Housekeeping

Genes

DNA template was obtained following the procedure as described in section

3.6.1. The PCR amplification and sequencing of partial 16S rRNA atpD (510 bp),

recA (530 bp) and glnII (600 bp) genes was performed as explained in section

earlier in section 3.6.2. Phylogenetic analysis was carried out as mentioned in

section 3.6.5.

3.11.3 Extraction of Bacterial DNA for DDH and G+C Determination

For determination of DNA-DNA hybridization (DDH) and G+C contents,

DNA of novel bacterial strain along with five reference strains was isolated

following the procedure illustrated by Marmur (1961) under the same lab

conditions. Bacterial cells were cultivated in TY medium. After centrifugation and

twice washing with distilled water, cells were suspended in 0.75 mL of 10 mM

Tris-HCL buffer (pH 8.0), and then added to 0.1 mL of the lytic solution

(Achromopeptidase 0.5 mg + lysozome 0.75 mg mL-1 of 10 mM Tris-HCL buffer,

pH 8.0), and put in incubator at 37 °C for 30 min. 70 µL of Tris-HCl buffer (1M

Tris-HCl, pH10.0 + SDS 10% (w/v)) and 10 µL of proteinase K (10 mg mL-1) were

added and incubated at 37 °C for 1-2 h. The cell mixture was freezing-thawing for

three times. Then, added 120 µL of 5M NaCl and 90 µL of CTAB/NaCl solution

(hexadecyltrimethyl ammonium bromide 10 g and NaCl 4.1 g 100 mL-1), mix

thoroughly and placed in an incubator at 65 °C for 20 min. The same amount of

67

phenol/chloroform/isoamylalchohol (25:24:1) was included, shaked vigorously for

10 min, followed by centrifugation at 10,000 rpm for 5 min and 0.4 mL of upper

phase was carefully taken out in another tube. The same amount of

chloroform/isoamylalchohol (24:1) was incorporated and shaken vigorously for 10

min. Samples were centrifuged at 10,000 rpm for 5 min, then 0.3 mL of upper

phase was taken into another tube and 50 µL of RNase solution (1 mg of RNase A

+ 400 units of RNase T1 mL-1 of 50 mM Tris HCl, pH 7.5) was included and the

solution was put in an incubator at 37 °C for 20 min. Phenol (0.2 mL, 90%, w/v)

and chloroform (0.2 mL) were added and mixed for 1 min. The centrifugation was

achieved at 10,000 rpm for 5 min, 0.3 mL of the supernatant was carefully taken

out in another tube. Ethanol (0.7 mL, 99%, w/v) was added and mixed very

slightly. Subsequently, DNA appeared in the solution was picked up using a glass

rod. Then DNA was washed in 70, 80 and 90% ethanol. After stepwise washes,

DNA was dried in an eppenpdorf tube under vacuum. The concentration and purity

of the isolated DNA was determined by using Beckman model DU-530

spectrophotomter. DNA solutions exhibiting a ratio of A260/A28 more than 1.8

were utilized for determining the DDH and G+C contents.

3.11.3.1 DNA-DNA hybridization

After DNA extraction, DNA-DNA hybridization was performed by

following the protocol of De Ley et al. (1970) by means of UV/VIS-

spectrophotometer equipped with a Peltier-thermostatted charger and a temperature

controller. Using ultrasonic, the DNA of bacterial samples were cut to fragments

having molecular weight around 2~5 X 105 D. Fragmented DNA was diluted with

the help of 0.1X SSC liquor to obtain concentration around A260= 1.5 ~ 2.0.

68

Wavelength of the spectrophotometer was adjusted at 260 nm. 300 µL sheared

DNA (A, B) was placed in two cuvettes separately. Put A, B each 150 µL in one

cuvette as sample M. A and B samples were put in boiling water for 15 min in

order to denature the DNA before measurement. One cuvette with 2X SSC was

used as a blank. Tor value was adjusted in the spectrophotometer. After reaching

Tor, the program for measuring DDH was run on spectrophotometer. A260 value

was recorded after every 1min till the nonlinear area was reached. Generally the

range of linear area is from 0~45 min, and 0~30 min was the measuring section.

Excel 5.0 was used to draw the straight line of renaturation, based on A260 against

time in order to get the regression equation. Slope of line was calculated to get

absolute value as the velocity of renaturation (expressed as V). The formula to

calculate DNA homology(H) is

H=[4VM-(VA+VB)]*100%/2 VBVA*

where VA,VB,VM are the renaturation velocity of A,B,M

In normal samples, VA or VB>VM>(VA+VB)/4

3.11.3.2 Determination of G+C contents

The G+C contents of novel bacteria and reference strains were calculated by

thermal denaturation technique illustrated by Marmur and Doty (1962).Escherichia

Coli K12 was included as a reference strain and the blank control was 0.1X SSC

solution. DNA concentration should be adjusted to A260=0.3-0.5. 300 µL DNA

was added in each cuvette. To estimate the melting temperature (Tm), 50 °C was

selected as the initial temperature, 95 °C as the final temperature, the climbing

speed was 1°C min-1 and A260 value was recorded per centigrade. Three

69

replications were run for each sample and the mean of two closest values was

selected as the final result. Following is the formula to calculate (G+C) is

(G+C) mol% = (Tm measured-Tm K12) x 2.08+51.2

Where Tm measured = Tm of tested strain;

Tm K12 = Tm of Escherichia Coli K12

3.11.4 Fatty Acid Profiling

Fatty acid methyl esters of novel nodule bacteria and five reference bacterial

species were extracted and analyzed by using Sherlock Microbial Identification

System (MIDI) and the procedure described by Sasser (1990). Bacterial strains

were cultured at 28 °C for about 1 week on TSA (Difco). The bottom part of a

clean tube was coated with cell biomass growing from the second, third and fourth

quadrant streaks using a 10 µL loop. It was saponified with 1 mL Sherlock Reagent

1, vortexed (10 sec) and boiled (5 min). It was then re-vortexed for another 10 sec,

boiled further for 25 min. The cooled mixture was methylated with 2 mL Sherlock

Reagent 2, vortexed and heated at 80 °C for 10 min and cooled rapidly. Methylated

fatty acids were extracted with the addition of 1.25 mL of Sherlock Reagent 3 and

gently mixed for 10 min. The resulting lower phase was pipetted and discarded.

The remaining upper phase was base washed with Sherlock Reagent 4 and mixed

for 5 min. Around 250 µL of saturated NaCl solution was added to clear the organic

phase (upper layer). Approximately 400 µL of the upper layer was transferred onto

a GC vial. The analysis of fatty acid was done by using the Hewlett Packard 5890

series II gas chromatograph equipped with an Ultra2 capillary column. The

microbial library was constructed and the similarity data were obtained by using the

Sherlock license CD v 6.0, TSBA6 database. Identification of fatty acids was

70

accomplished by evaluating the equivalent chain length (ECL) of single compound

to a peak naming table which covers more than 115 identified standards. The

measure of each fatty acid component was calculated as a percentage of the

complete fatty acid components contained within bacterium.

3.12 STATISTICAL ANALYSIS

The results complied for various characteristics were subjected to Analysis

of Variance and means was analyzed in completely randomized designed and

randomized completely block designed by taking treatments as the only factor

(Steel et al., 1997). Phylogenetic analyses were performed using bioinformatics

software like CLUSTAL X (version 1.8w), BioEdit and MEGA-5.1 packages

(Tamura et al., 2007).

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Chapter 4

RESULTS AND DISCUSSION

Extensive survey was carried out for collection of groundnut nodules and

rhizosphere soil from farmers field of pothwar, (Disrict Attock and Chakwal)

northern Punjab, Pakistan. The isolation of total 75 bacterial strains was

accomplished fromgroundnut nodules designated as BN-1, BN-2, BN-3……. and

rhizospheric soil designated as GS-1…..GS-27. These strains were evaluated for

their plant growth promoting (PGP) characterization including phosphate

solubilization, indole acetic acid (IAA) production and nifH gene amplification.

Identification was done by 16S rRNA gene sequencing. Housekeeping gene

sequence analysis for atpD, recA, and glnII genes of the strains identified as

Rhizobium was also performed to confirm their taxonomic position. Eight most

potential strains along with Rhizobium sp. were selected on the basis of their PGP

activities and tested for their ability to improve groundnut yield and N2-fixation

under controlled conditions. Three most potential strains were selected further on

the basis of their growth promoting performance under pot experiment and used in

field to investigate the integrated effect of PGPR along with full and half

recommended doses of chemical fertilizers.

4.1 CHARACTERIZATION OF BACTERIA FOR PLANT GROWTH

PROMOTING TRAITS

Forty seven bacterial strains were isolated from groundnut nodules and

thirtytwo bacterial strains were isolated from rhizospheric soil and grown on YMA

and TSA media, respectively. These bacterial strains were characterized for their

69

72

plant growth promoting attributes including indole-3-acetic acid (IAA) production,

phosphate solubilization, nifH gene amplification, bicochemical characterization

including catalase, oxidase, and Gram staining were carried out by following

manufacturer’s procedure (bioMérieux, France).

4.1.1 Phosphate Solubilization

The quantitative estimation of phosphate solubilized by isolated

strains is depicted in Table 8. The results revealed that all isolates were capable to

solubilize inorganic phosphate (tri-calcium phosphate), and the range was between

29.4 and 674.1 µg mL-1. The minimum quantity of insoluble phosphate was

solubilized by GS-27 (29.4 µg mL-1) and the maximum was solubilized by BN-44

(674.1 µg mL-1), followed by BN-55 (610.9 µg mL-1), GS-4 (562.3 µg mL-1), GS-6

(450.2 µg mL-1) and BN-5 (432.1 µg mL-1). The initial pH of the Nautiyal broth

was adjusted to 7.0 ± 0.05 which was reduced between 2.5 to 5.5 after 8 days of

incubation, indicating the ability of the isolates to synthesize organic acids

responsible for creating acidic conditions in the media. The drop in pH is associated

with the synthesis of organic acids by bacteria for example gluconic acid, α-2-

ketogluconic acid, lactic, isovaleric, isobutyric, acetic, malanoic and succinic acids

resulting in release of H+ ion (Hayat et al., 2010).

Phosphorous (P) is one of the primary nutrient element required for plant

developmentand obtaining optimum yield(Sajid et al., 2012). Some soil bacteria

have the ability to solubilze insoluble mineral phosphate and convert it into the

form available for the plants (Rodríguez et al., 2006). Bacteria belonging to genera

Pseudomonas, Rhizobium, Burkholderia, Bacillus, Arthrobacter and

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Achromobacter are widely used as phosphate solubilizing bacteria (PSB) in order to

improve crop yield and productivity as well as maintaining soil health since early

sixties (Khan et al., 2009b). Besides the production of organic acids, another

important aspect of P-solubilizing bacteria is the conversion of insoluble and

adsorbed forms of phosphorous into the soluble forms which plants can absorb

easily (Khan and Joergensen,2009). Son et al. (2006) has reported an increase in

nodule number, yield and nutrient uptake in soybean by the application of

Pseudomonas spp. The inoculation of phosphate solubilizing bacteria (PSB) also

accounted for an increased seedling length of chick pea(Sharma et al., 2007) and

enhance sugarcane yield (Sundara et al., 2002). There is 50% reduction of P

application without affecting yield with co-inoculation of PSB and PGPR in corn

(Yazdani et al., 2009), hence PSB can serve as efficient biofertilizer to improve

phosphorus availability to crop plants (Chen et al., 2006).

4.1.2 Indole-3-Acetic Acid (IAA) Synthesis

Seventy five isolated bacterial strains were screened for their ability to

synthesize IAA quantitatively. The bacterial strains were incubated in pure culture

(LB broth) in the presence (500 µg mL-1) and absence of L-tryptophan, added as a

precursor of IAA production (Table 8). Without tryptophan, the maximum

production of IAA was carried out by BN-55 (73.0 µg mL-1), followed by BN-5

and GS-6 which synthesized IAA in the range of 71.8 µg mL-1 and 49.2 µg mL-1,

respectively. Significant differences in IAA production were recorded with the

addition of tryptophan, with the maximum IAA production (97.6 µg mL-1) was

observed by GS-6, followed by BN-55 (95.9 µg mL-1) and BN-5 (80.1 µg mL-1),

indicating the ability of tryptophan to act as a precursor in IAA production. The

74

IAA production and release was stimulated by the addition of tryptophan, an amino

acid, signifying the dependence of IAA synthesis on tryptophan (Prasanna et al.,

2010). The average IAA synthesis by the bacterial strains was in the range of 9.5 µg

mL-1 to 97.6 µg mL-1, with tryptophan supplemented to the LB culture.

The ability to synthesize plant hormones is assumed to be a major quality of

more than 80% rhizospheric and symbiotic microorganisms (Hayat et al., 2010).

IAA is recognized as a key factor and considered as physiologically most active

auxin, which is directly beneficial for plants. IAA can activate the quick (e.g., cell

elongation) as well as long-term (i.e., cell division and differentiation) growth

processes in plants (Khan et al., 2009a). Increased number of root hairs and

increase in root length was observed in wheat crop by the addition of IAA (Datta

and Basu, 2000). The bacterial strain Pseudomonas putida has been found to

increase seedling root growth and enhance crop yield when it is used as innoculant

for tomato and canola crops (Patten and Glick, 2002). The PGPR like Bacillus spp.,

Enterobacter and Pseudomonas have been reported to increase root growth and

improve crop yield in field crops by solubilization of tri-calcium phosphate and

production of phytohormones (Khan et al., 2009b).

4.1.3 nifHgene amplification

The nitrogenase enzyme responsible for biological nitrogen fixation has a

dinitrogenase reductase subunit encoded by nifH gene (Halbleib and Ludden,

2000), which is used as the biomarker to study the ecology and evolution

ofnitrogen-fixing bacteria (Raymondet al., 2004). Symbiotic bacteria

75

includinggenus Rhizobium and many free living soil bacteria including

Azospirillum,

76

Table 8: Plant growth promoting traits of strains isolated from rhizospheric soil and nodules of groundnut.

S. No. Strain ID Location

IAA with tryptophan

IAA without

trytophan

P solubilization

Catalase Oxidase nif H amplification

Gram staining

----------------(µg mL-1)----------------

1. BN-1 Pindi Gheb 17.4±1.2 15±0.2 132 ±1.3 + + - + 2. BN-2 Pindi Gheb 41.3± 2.0 5.4±2.1 362 ± 4.0 + - - + 3. BN-3 Pindi Gheb 41.2±1.7 6.1±0.7 132 ±1.5 + + - - 4. BN-4 Pindi Gheb 20.9±1.5 9.6±1.0 199 ±5.2 + + - - 5. BN-5 Pindi Gheb 80.1±2.1 71.8±3.0 432±1.2 + + - + 6. BN-6 Pindi Gheb 10.9±1.5 20.5±1.1 152 ±1.3 + + + - 7. BN-7 Pindi Gheb 21.3±0.8 12.5±0.3 145 ±2.1 + + - - 8. BN-8 Pindi Gheb 20.0±3.1 9.3±0.8 81 ± 6.0 + - - + 9. BN-10 Pindi Gheb 26.7±2.4 9.3±1.6 54 ±4.2 + - - + 10. BN-12 Pindi Gheb 79.9±2.7 21.2±0.8 71 ±2.2 + - + - 11. BN-14 Kamra 34.8±1.8 23.0 ±1.6 54 ±1.2 + - - + 12. BN-15 Kamra 27.5±3.2 14.2±2.5 223 ±2.5 + - - 13. BN-16 Kamra 22.3±1.4 11.1±1.8 65 ±2.7 + + - - 14. BN-17 Kamra 31.5±1.7 14.2±1.9 88 ±3.4 + - - + 15. BN-18 Kamra 17.2±0.6 10.5±0.8 200 ±2.2 + + - - 16. BN-19 BARI 54.6±1.9 6.8±1.0 152±5.0 + + + - 17. BN-20 BARI 65.7±2.2 11.6±1.3 56 ±3.0 + - + - 18. BN-22 BARI 55.0±1.5 14.0 ±1.4 54 ±5.0 + - - - 19. BN-23 BARI 48.6±2.4 12.8±1.5 290 ±3.5 + - + - 20. BN-24 BARI 34.7±2.1 27.3±1.8 250 ± 5.0 + - - + 21. BN-25 BARI 15.0±1.7 8.3±1.6 58 ±3.8 + + - - 22. BN-26 BARI 18.9±1.4 12.2±1.2 63 ±4.2 + + - +

77

23. BN-27 BARI 19.4±1.0 8.0±2.0 155 ±2.7 + + - + 24. BN-28 Khunda 18.0 ±1.3 7.9±0.8 140 ±3.0 + - - + 25. BN-29 Khunda 22.1±1.6 11.2±1.3 33 ±2.9 + - - - 26. BN-30 Khunda 23.5±1.0 16.0 ±2.5 65 ± 3.9 + - - - 27. BN-31 Khunda 38.1 ±2.1 22.8±1.3 74± 2.7 + - - - 28. BN-32 Khunda 33.2±3.6 8.7±1.0 122 ±2.4 + - - - 29. BN-33 Khunda 32.2±1.5 16.1±0.8 76 ±2.8 + + - - 30. BN-38 Khunda 54.6±1.5 31.0 ±1.0 64 ±2.2 + + - - 31. BN-39 Koont 24.2±1.8 13.1±0.7 278 ± 5.6 + - + - 32. BN-41 Koont 13.7±1.3 6.5±0.5 153 ±3.0 + + - - 33. BN-42 Koont 54.5±2.1 13.0±1.1 195 ±3.2 + + - - 34. BN-43 Koont 25.2±1.3 11±0.6 100±6.8 + - - - 35. BN-44 Koont 44.5±2.1 23.1±1.3 674 ± 9.0 + - - - 36. BN-45 Koont 14.6± 1.6 10.6±0.6 147 ±2.6 + - - - 37. BN-46 Koont 25.5±2.5 10.1±0.7 342 ±5.2 + - - - 38. BN-47 Koont 21.1± 1.9 9.7±0.4 271±5.7 + - - - 39. BN-49 Koont 14.1±2.2 6.5±0.5 164 ±3.1 + - - + 40. BN-50 Koont 45.2±2.4 17±1.1 162 ±2.1 + + + - 41. BN-52 Fath-e-Jhung 30.4±2.1 14.4±0.7 253 ± 3.3 + - - - 42. BN-53 Fath-e-Jhang 32.4±0.4 8.4±0.1 125 ±2.1 + + - + 43. BN-55 Fath-e-Jhang 95.9±2.9 73.0±3 611 ± 3.0 + - - - 44. BN-56 Fath-e-Jhang 57.1±1.4 17.6±0.8 224 ±2.9 + - - - 45. BN-57 Dhudial 49.7±2.7 17.5±1.3 216 ± 2.1 + - - -

46. BN-58 Dhudial 29.6±4.3 11.3±1.3 130 ±4.2 + - - -

47. BN-59 Dhudial 57.1±2.6 19.6±1.6 191 ±2.8 + - - - 48. GS-1 BARI 10.5±1.0 10.3±0.7 190 ±1.7 + - - + 49. GS-2 BARI 57.6±1.2 27.3±0.7 301 ±3.8 + + - + 50. GS-3 BARI 32.3±1.0 24.5±1.4 130 ±1.8 + + - -

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51. GS-4 Koont 65.7±1.1 13.7±1.4 562 ±5.3 - + - + 52. GS-5 Koont 20.3±2.0 8.7±1.1 130±2.1 + + - + 53. GS-6 Koont 97.6±4.0 49.2±2.4 450±1.5 + - - + 54. GS-7 Koont 12.0 ±0.6 4.1±0.5 104 ±2.1 + - - - 55. GS-8 Koont 26.9±1.8 9.9±1.8 60 ±1.5 + - - + 56. GS-9 Koont 36.8±0.9 16.9±1.5 155 ±2.4 + + - + 57. GS-15 Koont 43.6±0.8 23.9±1.9 98 ±2.2 + - - - 58. GS-16 Koont 23.6±0.7 5.7±0.2 125 ±2.1 - + - - 59. GS-17 Koont 9.5±0.2 7.2±0.8 56 ±2.5 + + - + 60. GS-18 Koont 33.0±1.4 29.3±0.2 92 ±0.4 + + - + 61. GS-19 Ghazi Barotha 38.7±0.4 18.3±1.1 79 ±2.8 + + - - 62. GS-20 Ghazi Barotha 18.7±3.2 6.8±2.1 90 ±2.1 + + - - 63. GS-22 Ghazi Barotha 42.9±0.4 13.8±0.5 100 ±1.1 + - - + 64. GS-23 Ghazi Barotha 9.5±1.2 5.5±2.4 64 ±2.2 + - - - 65. GS-24 Ghazi Barotha 10.2±1.8 5.4±2.1 290 ±3.4 - - - - 66. GS-25 Ghazi Barotha 6.6±0.1 3.8±0.2 114 ±0.3 + + - + 67. GS-26 Ghazi Barotha 55.3±3.2 24.6±2.1 55 ±2.9 + + - + 68. GS-27 Ghazi Barotha 50.0±0.9 23.6±1.1 29 ±3.2 + + - + 69. GS-28 Ghazi Barotha 27.2±0.9 22.3±1.1 153 ±3.2 + + - - 70. GS-29 GRS, Attock 18.4±1.2 8.9±0.1 54 ±0.3 - - - - 71. GS-30 GRS, Attock 10.9±0.8 4.5±0.5 416 ±2.3 + + - + 72. GS-31 GRS, Attock 13.7±1.1 7.7±0.7 113 ±5.0 + + - - 73. GS-32 GRS, Attock 30.4±0.9 27.1±1.2 59 ±2.1 + + - - 74. GS-33 GRS, Attock 40.8±1.2 33.4±0.2 185 ±2.1 + + - +

79

75. GS-34 GRS, Attock 30.3±0.1 25.1±1.2 160 ±3.4 + + - +

+ Positive; - negative. Values indicate the mean ± SE for three replications. Burkholderia ferrariae FeGl01 and Burkholderia cepacia ATCC 25416are a standard reference strains for P-solubilization having the potential to solubilize inorganic P in the range of 97.7 and 88.2 µg mL-1,respectively (Inui-Kishiet al., 2012). Azotobacter vinelandii Mac 259 and Bacillus cereus UW 85 are a standard reference strains for IAA production having the potential to synthesize IAA in the range of 20.9 and 40.2µg mL-1,respectively (Paterno, 1997).

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Azotobacter, Klebsiella,Rhodospirillum,Azotobacter, Clostridium, Azospirillum,

Burkholderia, Azoarcusand Rhodobacterhave been reported to possess nifH gene

and are involved in the process of N2-fixation, thus act as PGPR (Franche et al.,

2009). The nifH gene phylogeny is partly congruent with that of 16S rRNA gene

and is suitable for N2-fixing bacteria identification at species or genus level.

Moreover, nifH gene sequence balance could provide new insights into the genetic

diversity of N2-fixing bacteria (Gaby and Buckley,2014).

DNA of all the bacterial strains isolated from groundnut rhizospheric soil

and root nodules was amplified for nifH gene (400 bp). Nitrogen fixing ability was

coded in 8 strains isolated from root nodules: BN-6, BN-12, BN-19, BN-20, BN-

23, BN-25, BN-39 and BN-50. In order to minimize the harmful effect of chemical

fertilizers, use of PGPR that have the ability of N2-fixation should be encouraged to

facilitate optimum crop yield, without deteriorating soil health.

4.1.4 Gram Staining and Catalase, Oxidase Activity

The results for Gram staining, catalase and oxidase activities for all tested

isolates are depicted in Table 8. Gram staining is one of the oldest methods of

bacterial taxonomy and is still a valid minimal criterion for bacterial identification

(Tindal et al., 2010). The isolated bacterial strains belonged to genus Bacillus,

Arthrobacter, Microbacterium, Curtobacterium and Kocuria were Gram positive,

whereas the strains from genus Pantoea, Pseudomonas, Rhizobium,

Flavobacterium, Acinetobacter, Erwinia, Enterobacter, Escherichia, Kosakonia,

Stenotrophomonas, Variovorax and Chryseobacterium were Gram negative.

Catalase is a common enzyme responsible for the catalyses of hydrogen peroxide

81

into hydrogen and oxygen, hence protecting the cell from oxidative damage

(Chelikani et al., 2004), whereas oxidase enzyme is involved in oxidation-reduction

reaction in which oxygen is electron acceptor. Of all the 75 tested bacterial strains,

71 were positive for catalase production. Strains GS-3, GS-16, GS-24 and GS-29

were negative for catalase activity; and 37 strains had the ability to synthesize

oxidase.

4.2 MOLECULAR IDENTIFICATION OF BACTERIA

4.2.1 16S rRNA Gene Sequencing

The comparative analysis of small subunit ribosomal RNA (16S rRNA)

gene sequence is recognized as the most widely used classification techniques in

prokaryotic identification and systematic (Woese, 1992), and has emerged as one

of the reliable universal phylogenetic markers linked to microbial plankton

identification and naming (Giovannoni and Rappé, 2000). Identification of fifty

four potential PGPR isolated from groundnut rhizospheric soil and nodules was

performed using molecular technique of 16S rRNA gene sequencing. Nearly

complete gene sequence results of each strain was checked, edited and aligned by

using BioEdit ver. 7.1.3.0 to obtain consensus sequence. The obtained sequences

were BLAST in EzBiocloud database (http://ezbiocloud.net/eztaxon) to obtain

sequence percent similarities and possible identities with their closely related

species. All identified strains exhibited more than 97% similarity with the closely

validated species. The strains belonged to 17 different genera including Bacillus,

Pantoea, Pseudomonas, Arthrobacter, Rhizobium, Microbacterium,

Flavobacterium, Acinetobacter, Kosakonia, Curtobacterium, Variovorax,

Stenotrophomonas, Erwinia, Escherichia, Enterobacter, Chryseobacterium and

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Kocuria (Table 9), demonstrating the diversity of bacterial strains isolated from

different locations. Only one strain isolated from groundnut nodules, BN-19,

identified as Rhizobium sp. exhibited 97.5% similarity with its closely related

strains; thus has the potential to be a novel and validated as a novel Rhizobium sp.

by adopting minimal criteria of species identification (including housekeeping gene

sequencing, DNA-DNA hybridization, fatty acid profiling, BIOLOG and

phenotypic characterization).

The nearly complete 16S rRNA sequences of each identified bacteria were

submitted and registered online at the DNA Data Bank of Japan (DDBJ) using the

Sakura Nucleotide Data Submission System to obtain Accession Number of each

sequence (Table 9).

4.2.2 Housekeeping genes sequencing

Currently, the housekeeping gene analysis of multiple protein-encoding

genes has become an extensively applied method for the confirmation of taxonomic

relationships between species of same genus (Wertz et al., 2003). As compared to

16S rRNA genes, multilocus sequence analysis (MLSA) is more reliable tool for

identification purposes, since the 16S rRNA gene sequences usually do not

accounts for species differentiation due to its more-conserved nature (Case et al.,

2007). According to Zeigler (2003), MLSA of small number of carefully selected

genes including atpD, dnaK, glnII, glnA, gltA, gyrB, recA, rpoBand thrCcan yield

sequence clusters at an extensive range of taxonomic levels, from intra-specific

through the species level to clusters at higher levels.

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Table 9: Identification of bacterial strains isolated from groundnut based on 16S rRNA gene sequencing and their assigned accession

numbers.

Sr. # Strain ID Location Source of isolation

Closely related Taxa identified by BLAST search using ezbiocloud.net

Species Highest similarity of

16SrRNA gene sequence % DDBJ Accession

1 BN-2 Pindi Gheb Nodules Bacillus safensis 100 AB971370

2 BN-3 Pindi Gheb - Pantoea agglomerans 99.6 AB971371

3 BN-4 Pindi Gheb - Pseudomonas oryzihabitans 100 AB971372

4 BN-5 Pindi Gheb - Arthrobacter globiformis 98.7 AB971373

5 BN-6 Pindi Gheb - Rhizobium alkalisoli 99.7 AB969782

6 BN-8 Pindi Gheb - Microbacterium oryzae 99.13 LC038163

7 BN-10 Pindi Gheb - Bacillus sp. 100 LC038164

8 BN-12 Pindi Gheb - Rhizobium massiliae 100 AB969783

9 BN-14 Kamra - Bacillus tequilensis 99.48 LC038165

11 BN-16 Kamra - Flavobacterium oceanosedimentum 99.21 LC038166

12 BN-17 Kamra - Bacillus simplex 98.47 LC038167

13 BN-19 BARI - Rhizobium loessense 97.5 AB854065

14 BN-20 BARI - Rhizobium huautlense 99.9 AB969784

15 BN-22 BARI - Acinetobacter junii 98.9 AB777646

16 BN-23 BARI - Rhizobium pusense 99.9 AB969785

17 BN-24 BARI - Microbacterium natoriense 99.3 LC040930

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18 BN-25 BARI - Rhizobium herbae 99.3 AB969786

19 BN-26 BARI - Bacillus subtilis 99.9 LC040931

20 BN-27 BARI - Enterobacter cloacae 100 LC040932

21 BN-28 Khunda - Bacillus simplex 99 LC040933

22 BN-29 Khunda - Kosakonia cowanii 99.33 LC040934

23 BN-30 Khunda - Kosakonia cowanii 99.6 LC040935

24 BN-31 Khunda - Pseudomonas azotoformans 99.7 LC040936

25 BN-32 Khunda - Pseudomonas azotoformans 99.7 LC040937

26 BN-33 Khunda - Curtobacterium plantarum 98.4 LC040938

27 BN-38 Khunda - Pseudomonas sp. 99.5 LC040939

28 BN-39 Koont - Rhizobium massiliae 100 AB969787

29 BN-41 Koont - Variovorax paradoxus 99 LC040940

30 BN-43 Koont - Pseudomonas sp. 100 LC040941

31 BN-44 Koont - Pseudomonas oryzihabitans 100 LC040942

32 BN-45 Koont - Erwinia persicinus 99.22 LC040943

33 BN-46 Koont - Pseudomonas oryzihabitans 100 LC040944

34 BN-47 Koont - Pseudomonas sp. 100 AB773830

35 BN-49 Koont - Bacillus sp. 99.41 LC040945

36 BN-50 Koont - Rhizobium alkalisoli 99.77 AB969788

37 BN-52 Fath-e-Jhung - Escherichia vulneris 99.27 LC040946

85

38 BN-55 Fath-e-Jhang - Pseudomonas psychrotolerans 100 LC040947

39 BN-56 Fath-e-Jhang - Stenotrophomonas maltophilia 99.11 LC040948

40 BN-57 Dhudial - Stenotropomonas malotphilia 99.19 LC040949

41 BN-58 Dhudial - Enterobacter cloacae 99.85 LC040950

42 BN-59 Dhudial - Stenotropomonas maltphilia 99.11 LC040951

48 GS-1 BARI Soil Bacillus subtilis 99.82 AB773829

49 GS-2 BARI Soil Kocuria turfanensis 99.19 LC040954

50 GS-3 BARI Soil Chryseobacterium arthrosphaerae 98.75 LC040953

51 GS-4 Koont Soil Bacillus aryabhattai 99.09 LC040952

52 GS-5 Koont Soil Bacillus oceanisediminis 97.98 LC040955

53 GS-6 Koont Soil Microbacterium arborescens 100.0 LC057387

54 GS-7 Koont Soil Acinetobacter pittii 100.0 AB777645

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Eight bacterial strains namely BN-6, BN12, BN-19, BN-20, BN-23, BN-25,

BN-39 and BN-50 isolated from groundnut nodules were placed in genes

Rhizobium after BLAST search results of 16S rRNA gene sequence. A polyphasic

study including multi-gene amplification was carried out to elucidate their

taxonomic position in genus Rhizobium. Housekeeping gene sequencing of atpD,

recA and glnII genes was performed to confirm the phylogenetic status of the

above mentioned strains (Table 10). The sequence obtained were BLAST using

NCBI database and the results confirmed more than 96% similarity of the seven

strains except BN-19 with closely related identified Rhizobium species. The strain

BN-19 exhibited less than 94% sequence similarity with the closely related

identified Rhizobium species, hence proved its potential to be a novel candidate

(Section 4.7). Housekeeping gene sequence of three genes were submitted in DDBJ

and Accession Numbers of the genes were acquired (Table 10).

4.2.3 Phylogenetic Analysis of 16S rRNA and Housekeeping Genes

A phylogenetic tree, phylogeny or dendogram illustrates the lines of

evolutionary lineage of various species, organisms or genes representing mutual

ancestor (Baum, 2008). When it comes to the phylogenetic analysis, it is inevitable

to mention the theory of evolution, the stepping stone of phylogenies (Maddison

and Maddison, 1992). Every node with descendants in the tree signifies the most

recent common ancestor exhibiting edge lengths equivalent to time approximates.

In tree, the single node is called the taxonomic unit. An important step in the

phylogeny construction is to check the sequences and to align them. Aligned

sequence positions subjected to phylogenetic analysis represent a prior

87

phylogenetic conclusion because the sites themselves are effectively related

genealogically.

In the present research, the 16S rRNA gene sequences of the isolated strains

are compared with the sequence retrieved from GenBank (EzBiocloud), and

housekeeping gene sequence alignment was carried out with the help of ClustalW

software. Neighbor-joining tree of each genus was constructed (Figure 7-21), and

the results depicted that each identified bacterial species lies in the subclade of

closely identified species. For housekeeping genes of the Rhizobium sp., sequences

were obtained from NCBI GenBank, and neighbor-joining trees clarifying the

positions of each strain in the genus were constructed (Figure 22-24).

4.3 BIOCHEMICAL AND PHENOTYPIC CHARACTERIZATION OF

Rhizobium sp.

The bacterial strains BN-6, BN-12, BN-19, BN-20, BN-23, BN-25, BN-39

and BN-50 were identified as belonging to genus Rhizobium after 16S rRNA and

housekeeping genes sequence analysis. Their biochemical characterization was

performed keeping in view their ability to utilize different carbon and energy

sources and for this purpose Biolog GN-2 miocroplates were used to generate a

metabolic fingerprinting of bacterial strains. The method comprises of a 96 well

plate, with 95 different carbon sources and a blank well.Even though initially

Biolog was used for identification of clinical bacteria, but now it is extensively

used for classification, categorization, and determination of diversity studies of

different microorganisms (Emerson et al., 2008). Carbon source utilization can

determine the ability of an organism to live symbiotically or saprophytically in the

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Table 10: Sequence similarities (%) and accession numbers for atpD, recA and glnII genes between Rhizobiumsp.and reference

strains.

Strain ID 16S rRNA gene

identification atpD recA glnII DDBJ Accession Number

atpD recA glnII BN-6 R. alkalisoli 95.5 99.1 100 LC061200 AB971367 AB971366 BN-12 R. massiliae 97.8 99.4 99.1 LC061201 LC061206 LC061211 BN-19 R. loessense 93.3 89.5 89.0 AB856324 AB855792 AB856325 BN-20 R. huautlense 100 89.2 99.2 AB970799 AB970801 AB970800 BN-23 R. pusense 98.2 99.6 100 LC061202 LC061207 LC061212 BN-25 R. herbae 100 100 98.7 LC061203 LC061208 LC061213 BN-39 R. massiliae 95.7 99.5 99.6 LC061204 LC061209 LC061214 BN-50 R. alkalisoli 100 99.7 99.4 LC061205 LC061210 LC061215

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GS-5(LC040955)

GS-1 (AB773829)

Bacillus subtilis subsp. spizizenii NRRL B-23049T (CP002905)

Bacillus tequilensis KCTC 13622T (AYTO01000043)

BN-10 (LC038164),BN-14 (LC038165), BN-49(LC040945)

Bacillus subtilis subsp. subtilis NCIB 3610T (ABQL01000001)

BN-26 (LC040931)

Bacillus amyloliquefacienssubsp. plantarum FZB42T (CP000560)

Bacillus siamensis KCTC 13613T (AJVF01000043)

Bacillus amyloliquefaciens subsp. amyloliquefaciens DSM 7T (FN597644)

Bacillus atrophaeus JCM 9070T (AB021181)

BN-2 (AB971370), BN-17(LC038167), BN-28(LC040933)

Bacillus safensis FO-36bT (ASJD01000027)

Bacillus pumilus ATCC 7061T (ABRX01000007)

Bacillus invictus Bi.FFUP1T (JX183147)

Bacillus stratosphericus 41KF2aT (AJ831841)

Bacillus altitudinis 41KF2bT (ASJC01000029)

Bacillus aerophilus 28KT (AJ831844)

Bacillus xiamenensis HYC-10T (AMSH01000114)

Brevibacillus brevis (D78457)

50

52

48

74

55

56

99

76

62

88

99

100

0.01

Figure 7: Neighbour-joining phylogenetic tree of strains BN2, BN-10, BN-14, BN-17, BN-26, BN-28, BN-49, GS-1 and GS-5

based on nearly complete 16S rRNA gene sequencing, clearly depicting their taxonomic status as belonging to genus Bacillus.

86

90

Figure 8: A phylogenetic tree (16S rRNA gene sequence) of strain BN-3 constructed by neighbor-joining method. Bootstrap values

>50% are indicated at the nodes. The sequence of Pectobacterium carotovorum (AJ233411) was used as outgroup.

Pantoea vagans LMG 24199T (EF688012)

Pantoea brenneri LMG 5343T (EU216735)

Pantoea conspicua LMG 24534T (EU216737)

BN-3 (AB971371)

Curtobacterium plantarum CIP 108988T (JN175348)

Pantoea agglomerans DSM 3493T (AJ233423)

Pantoea eucalypti LMG 24198T (EF688009)

Pantoea anthophila LMG 2558T (EF688010)

Pantoea deleyi LMG 24200T (EF688011)

Pantoea ananatis LMG 2665T (JMJJ01000010)

Flavobacterium acidificum LMG 8364T (JX986959)

Pantoea allii LMG 24248T (AY530795)

Pantoea stewartii subsp. stewartii LMG 2715T (Z96080)

Pantoea stewartii subsp. indologenes LMG 2632T (Y13251)

Pantoea septica LMG 5345T (EU216734)

Pectobacterium carotovorum (AJ233411)

63

73

55

99

99

74

0.005

87

91

BN-31 (LC040936) BN-32 (LC040937), BN-38 (LC040939)

Pseudomonas azotoformans IAM1603T (D84009)

Pseudomonas fragi ATCC 4973T (AF094733)

Pseudomonas chengduensis MBRT/EU307111

Pseudomonas toyotomiensis HT-3T (AB453701)

Pseudomonas oleovorans subsp. lubricantis RS1T (DQ842018)

Pseudomonas tuomuerensis 78-123T (DQ868767)

Pseudomonas stutzeri ATCC 17588T (CP002881)

Pseudomonas benzenivorans DSM 8628T (FM208263)

Pseudomonas flavescens B62T (U01916)

Pseudomonas luteola IAM 13000T (D84002)

BN-46(LC040944)

BN-43 (LC040941)

BN-44 (LC040942)

Pseudomonas oryzihabitans IAM 1568T (AM262973)

BN-55(LC040947)

BN-47(AB773830)

BN-4 (AB971372)

Pseudomonas psychrotolerans C36T (AJ575816)

Acenitobacter lwoffii (U10875)

100

99

66

100

36

40

100

0.01

Figure 9:Neighbour-joining tree of strains BN-4, BN-31, BN-32, BN-38, BN-43, BN-44, BN-46, BN-47 and BN-55 showing

the phylogenetic relationships of therepresentative strains with the closely related strains of genus Pseudomonas.

88

92

Arthrobacter polychromogenes DSM 20136T (X80741)

Arthrobacter oxydans DSM 20119T (X83408)

Arthrobacter scleromae YH-2001T (AF330692)

Arthrobacter siccitolerans 4J27T (GU815139)

Arthrobacter phenanthrenivorans Sphe3T (CP002379)

Arthrobacter niigatensis LC4T (AB248526)

Arthrobacter defluvii 4C1-aT (AM409361)

Arthrobacter chlorophenolicus A6T (CP001341)

Arthrobacter equi IMMIB L-1606T (FN673551)

Arthrobacter enclensis NIO-1008T (JF421614)

BN-5 (AB971373)

Arthrobacter oryzae KV-651T (AB279889)

Arthrobacter humicola KV-653T (AB279890)

Arthrobacter globiformis NBRC 12137T (BAEG01000072)

Arthrobacter pascens DSM 20545T (X80740)

Arthrobacter flavus JCM 11496T(AB299278)

Arthrobacter crystallopoietes DSM 20117T (X80738)

Psudomonas putidia (AJ007910)

77

99

62

82

87

54

65

88

100

99

78

60

71

0.02

Figure 10: Neighbour-joining tree of strain BN-5 based on 16S rRNA gene sequence with closely related identified species of

genus Arthrobacter. Bootstrap values > 50% are shown at the nodes.Psudomonas putidia is used as outgroup.

89

93

BN-12 (AB969783)Rhizobium pusenseNRCPB10T(FJ969841)BN-23 (AB969785)BN-39 (AB969787)

Rhizobium vitis LMG 8750T(X67225)BN-25 (AB969786)

Rhizobium herbae CCBAU 83011T(GU565534) Rhizobium leguminosarum LMG 14904T(AM181757)Rhizobium etli CFN 42T(U28916)Rhizobium phaseoli ATCC 14482T(EF141340)

Rhizobium pakistanensisBN-19T(AB854065)Rhizobium tarimensePL-41T(HM371420)Rhizobium soliDS-42T(EF363715) Rhizobium cellulosilyticumALA10B2T(DQ855276)

Rhizobium alkalisoli CCBAU 01393T (EU074168)Rhizobium vignae CCBAU 05176T(GU128881)BN-20 (AB969784)Rhizobium huautlense S02T(AF025852)BN-50 (AB969788)

BN-6(AB969782)Rhizobium oryzae Alt 505T (EU056823)

Rhizobium pseudoryzaeJ3-A127T(DQ454123)Bradyrhizobium japonicum USDA 6T (NC017249)

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43

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71

42

62

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92

32

19

43

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0.01

Figure 11: Neighbour-joining tree of isolated Rhizobium strains on the basis of nearly complete 16S rRNA gene sequence,

indicating the position of the isolated strains in genus Rhizobium. Bradyrhizobium japonicum USDA 6T was used as outgroup.

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94

BN-8 (LC038163)

Microbacterium barkeri DSM 20145T (X77446)

Microbacterium indicum BBH6T (AM158907)

Microbacterium oryzae MB10T (GU564360)

Microbacterium luticocti DSM 19459T (AULS01000007)

Microbacterium immunditiarum SK 18T (DQ119293)

Microbacterium marinilacus YM11-607T (AB286020)

Microbacterium paludicola US15T (AJ853909)

Microbacterium radiodurans GIMN1.002T (GQ329713)

Microbacterium arborescens DSM 20754T (X77443)

Microbacterium natoriense TNJL143-2T (AY566291)

BN-24(LC040930)

Microbacterium azadirachtae AI-S262T (EU912487)

Microbacterium kyungheense THG-C26T (JX997973)

Microbacterium marinum H101T (HQ622524)

Microbacterium arthrosphaerae CC-VM-YT (FN870023)

Microbacterium jejuense THG-C31T (JX997974)

Leucobacter aerolatus(FN597581)

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63

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83

69

0.005

Figure 12: A phylogenetic tree of strains BN-8, BN-24 and GS-6 constructed on the basis of 16S rRNA gene sequence by

neighbor-joining method. The tree is defining the taxonomic position of the isolated strain in genus Microbacterium.

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95

Figure 13: Dendogram of BN-16 derived from 16S rRNA gene sequence constructed by neighbor-joining method.

Brevibacterium linens DSM 20425T was used as outgroup.

Curtobacterium albidum DSM 20512T (AM042692)Curtobacterium citreum DSM 20528T (X77436)

Flavobacterium oceanosedimentum ATCC 31317T (EF592577) BN-16 (LC038166)Curtobacterium pusillum DSM 20527T (AJ784400)

Curtobacterium luteum DSM 20542T (X77437)Curtobacterium ammoniigenes NBRC 101786T (AB266597)

Curtobacterium herbarum P 420/07T (AJ310413)Curtobacterium flaccumfaciens LMG 3645T (AJ312209)

Mycetocola tolaasinivorans CM-05T (AB012646) Mycetocola lacteus CM-10T (AB012648)

Compostimonas suwonensis SMC46T (JN000316) Frondihabitans sucicola GRS42T (JX876867)

Brevibacterium linensDSM 20425T (X77451)

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45

40

32

30

47

36

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0.01

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96

Acinetobacter johnsonii CIP 64.6T (APON01000005)

Acinetobacter oryzae B23T (GU954428)

Acinetobacter bouvetii DSM 14964T/APQD01000004

Acinetobacter kyonggiensis KSL5401-037T (FJ527818)

Acinetobacter beijerinckii CIP 110307T (APQL01000005)

Acinetobacter kookii 11-0202T (JX137279)

Acinetobacter gyllenbergii CIP 110306T (ATGG01000001)

Acinetobacter parvus DSM 16617T (AIEB01000124)

Acinetobacter tjernbergiae DSM 14971T (ARFU01000016)

Acinetobacter venetianus RAG-1T (AKIQ01000085)

Prolinoborus fasciculus CIP 103579T (JN175353)

GS-7 (AB777645)

Acinetobacter pittii (HQ180184)

Acinetobacter indicus CIP 110367T (KI530754)

BN-22 (AB777646)

Acinetobacter junii CIP 64.5T (APPX01000010)

Acinetobacter baumannii ATCC 19606T (ACQB01000091)

Acinetobacter gerneri DSM 14967T (APPN01000041)

Psychrobacter immobilis (U39399)

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53

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100

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67

62

0.01

Figure 14: Neighbour-joining tree illustrating the relationship of BN-22 and GS-7 with closely related Acinetobacter species.

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Figure 15: Phylogenetic tree obtained by neighbor-joining method indicating the relationship between the strains BN-29

and BN-30 and the closely related taxa. Sodalis glossinidius M1T was used as outgroup.

Escherichia coli O157 EC4115T (CP001164)

Escherichia fergusonii ATCC 35469T (CU928158)

Cedecea lapagei GTC 346T (AB273742)

Citrobacter farmeri CDC 2991-81T (AF025371)

Salmonella enterica subsp. enterica LT2T (AE006468)

Salmonella enterica subsp. salamae DSM 9220T(EU014685)

Enterobacter cloacae subsp. cloacae ATCC 13047T (CP001918)

Escherichia hermannii GTC 347T (AB273738)

Kosakonia cowanii CIP 107300T (AJ508303)

BN-30 (LC040935)

BN-29 (LC040934)

Kosakonia oryzae Ola 51T (EF488759)

Enterobacter hormaechei ATCC 49162T (AFHR01000079)

Enterobacter ludwigii DSM 16688T (AJ853891)

Sodalis glossinidius M1T (M99060)

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41

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84

50

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0.005

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98

Figure 16: Unrooted phylogenetic tree of strain BN-33 based on 16S rRNA gene sequence. The tree was constructed by

neighbor-joining method. Brevibacterium linens DSM 20425T was used as outgroup.

BN-33 (LC040938)Pantoea conspicua LMG 24534T (EU216737)Curtobacterium plantarum CIP 108988T (JN175348) Pantoea anthophila LMG 2558T (EF688010)Pantoea vagans LMG 24199T (EF688012)Pantoea agglomerans DSM 3493T (AJ233423)Pantoea brenneri LMG 5343T (EU216735)Pantoea deleyi LMG 24200T (EF688011)

Pantoea ananatis LMG 2665T (JMJJ01000010) Pantoea stewartii subsp. indologenes LMG 2632T (Y13251)Pantoea stewartii subsp. stewartii LMG 2715T (Z96080) Pantoea allii LMG 24248T (AY530795)Flavobacterium acidificum LMG 8364T (JX986959)Pantoea eucalypti LMG 24198T (EF688009)

Pantoea gaviniae A18/07T (GQ367483)Pantoea septica LMG 5345T (EU216734)Pantoea rwandensis LMG 26275T (JF295055)

Brevibacterium linensDSM 20425T (X77451)

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91

24

23

28

43

40

41

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0.02

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99

Figure 17: Neighbour-joining phylogenetic tree confirming the position of strain BN-45 in genus Erwinia. Brenneria

alni ICMP 12481T was used as outgroup.

Erwinia amylovora NBRC 12687T (BAYW01000035)Erwinia pyrifoliae DSM 12163T (FN392235)Erwinia uzenensis YPPS 951T (AB546198)

Erwinia piriflorinigrans CFBP 5888T (GQ405202) Erwinia tasmaniensis Et1/99T (CU468135)

Erwinia rhapontici ATCC 29283T/U80206Candidatus Erwinia dacicolaBactrocera oleae (AJ586620)

BN-45 (LC040943)Erwinia persicina ATCC 35998T (U80205)

Erwinia billingiae Eb661T (AM055711)Erwinia toletana CECT 5263T (FR870447) Pantoea eucrina LMG 2781T (EU216736)

Pantoea wallisii LMG 26277T (JF295057)Pantoea rodasii LMG 26273T (JF295053)

Pantoea rwandensis LMG 26275T (JF295055)Pantoea septica LMG 5345T (EU216734)

Brenneria alniICMP 12481T (AJ223468)

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85

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81

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52

33

39

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58

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0.005

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100

Figure 18: Dendogram of BN-52 derived from 16S rRNA gene sequence constructed by neighbor-joining method.

Enterobacter cancerogenus LMG 2693T (Z96078) Enterobacter asburiae JCM 6051T (AB004744)

Enterobacter ludwigii DSM 16688T (AJ853891) Leclercia adecarboxylata GTC 1267T (AB273740)

Enterobacter xiangfangensis 10-17T (HF679035)Enterobacter hormaechei ATCC 49162T (AFHR01000079)

BN-52 (LC040946)Escherichia vulneris ATCC 33821T (AF530476)Klebsiella michiganensis W14T (JQ070300)Klebsiella oxytoca JCM 1665T (AB004754)

Enterobacter aerogenes KCTC 2190T (CP002824)Yokenella regensburgei GTC 1377T (AB273739)

Citrobacter freundii ATCC 8090T (ANAV01000046)Citrobacter murliniae CDC 2970-59T (AF025369)

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88

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40

12

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30

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0.001

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Leclercia adecarboxylata GTC 1267T (AB273740)

Enterobacter ludwigii DSM 16688T (AJ853891)

Enterobacter kobei CIP 105566T (AJ508301)

Enterobacter cloacae subsp. dissolvens LMG 2683T (Z96079)

BN-58 (LC040950)

Enterobacter hormaechei ATCC 49162T (AFHR01000079)

Erwinia aphidicola DSM 19347T (AB273744)

Citrobacter freundii ATCC 8090T (ANAV01000046)

Citrobacter murliniae CDC 2970-59T (AF025369)

Citrobacter youngae CECT 5335T (AJ564736)

Salmonella enterica subsp. enterica LT2T (AE006468)

Enterobacter cloacae subsp. cloacae ATCC 13047T (CP001918)

Klebsiella oxytoca JCM 1665T (AB004754)

Klebsiella michiganensis W14T (JQ070300)

Cedecea lapagei GTC 346T (AB273742)

Enterobacter oryzendophyticus REICA 082T (JF795011)

Buchnera aphidicola (M63246)

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29

70

79

61

30

33

9

95 30

34

66

0.01

Figure 19: Phylogenetic relationship of the strain BN-58 with its closely related species on the basis of aligned sequences of

16S rRNA genes.

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102

Figure 20: Phylogenetic relationship of GS-3 on the basis of 16S rRNA gene within the genus Chryseobacterium.

Significant bootstrap levels are indicated at the nodes.

Chryseobacterium vietnamense GIMN1.005T (HM212415)

Chryseobacterium bernardetii NCTC 13530T (JX100816)

Chryseobacterium nakagawai NCTC 13529T (JX100822)

Chryseobacterium aquifrigidense CW9T (EF644913)

GS-3 (LC040953)

Chryseobacterium gleum ATCC 35910T (ACKQ01000057)

Chryseobacterium arthrosphaerae CC-VM-7T (FN398101)

Chryseobacterium indologenes NBRC 14944T(BAVL01000024)

Chryseobacterium tructae 1084-08T (FR871429)

Chryseobacterium contaminans C26T (KF652079)

Chryseobacterium artocarpi UTM-3T (KF751867)

Chryseobacterium oncorhynchi 701B-08T (FN674441)

Chryseobacterium viscerum 687B-08T (FR871426)

Chryseobacterium lactis NCTC 11390T (JX100821)

Chryseobacterium oranimense H8T (EF204451)

Flavobacterium aquatile (JX657053)

98 52

63

30

6

60

8

61

86

43

94

0.02

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103

Figure 21: Neighbour-joining phylogenetic tree constructed on the basis of 16S rRNA gene sequences depicting the

relationship of GS-2 with closely realted species of genus Kocuria. Micrococcus luteus (AJ536198) was used as

outgroup.

Kocuria himachalensis K07-05T (AY987383)

Kocuria rosea DSM 20447T (X87756)

Kocuria polaris CMS 76T (AJ278868)

Kocuria aegyptia YIM 70003T (DQ059617)

Kocuria sediminis FCS-11T (JF896464)

GS-2(LC040952)

Kocuria turfanensis HO-9042T (DQ531634)

Kocuria flava HO-9041T (EF602041)

Kocuria marina KMM 3905T (AY211385)

Kocuria gwangalliensis SJ2T (EU286964)

Kocuria atrinae P30T (FJ607311)

Kocuria assamensis S9-65T (HQ018931)

Kocuria palustris DSM 11925T (Y16263)

Micrococcus luteus (AJ536198)

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81 95

48

70

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0.005

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Figure 22: Phylogenetic tree of isolated Rhizobium strains based on housekeeping gene sequence of atpD gene.

BN-20 (AB970799) BN-50 (LC061205)

BN-25(LC061203)

BN-12 (LC061201)

R. huautlense (AM418782)

BN-39 (LC061204)

BN-6 (LC061200)

R. pakistanensisBN-19T (AB856324)

BN-23 (LC061202)

R. alkalisoliLMG24763T(EU672461)

R. galegae (AM418779)

R. cellulosilyticum (AM286429)

R. soliDS-42T (GQ260191)

R. yanglingenseSH22623T(AY907373)

R. multihospitium CCBAU 83401T(EF490019)

R. tropici LMG 9503T(AM418789)

R. etli CFN 42T (CP000133)

R. fabae CCBAU 33202T(EF579929)

R. leguminosarum (AJ294405)

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100

80

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52

45

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0.01

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105

Figure 23: Phylogenetic tree of isolated Rhizobium strains based on housekeeping gene sequence of recA gene

constructed by neighbor-joining method.

BN-50 (LC061210) BN-6 (AB971367) BN-25 (LC061208)

R. alkalisoliLMG24763T(EU672490)R. huautlense (AM182128)

R. giardinii (AM182123)R. galegae (AM182127)

R. soliDS-42T (GQ260192) R. etli CFN 42T

R. fabae CCBAU 33202T(EF579941)R. leguminosarum USDA 2370T (AJ294376)

R. cellulosilyticum LMG 23642T(AM286427)R. tropici A USDA 9039T(AJ294372)

R. tropici B USDA 9030T(AJ294373)

R. pakistanensisBN-19T(AB855792) BN-20 (AB970801

BN-23 (LC061207) BN-12 (LC061206 ) BN-39 (LC061209)

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106

Figure 24: Phylogenetic tree of isolated Rhizobium strains based on housekeeping gene sequence of glnII gene

constructed by neighbor-joining method.

BN-25 (LC061213)

BN-6 (AB971366)

BN-50 (LC061215) R. alkalisoliLMG24763T(EU672475)

BN-20 (AB970800)

R. gallicum (AY929427)

BN-23 (LC061212)

BN-39 (LC061214)

BN-12 (LC061211) R. pakistanensis BN-19T (AB856325)

R. etli (AF169585)

R. leguminosarum (EU155089)

R. galegae (AF169587)

R. lusitanum (EF639841)

R. tropici (AF169583)R multihospitium CCBAU 83401T (EF490040)

R. tropici (AF169584)

S. xinjiangense (EU155088)

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100 47

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24

70 33

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71

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107

soil(Brückner and Titgemeyer, 2002). Because of its low cost and ease of usage, it

is useful for large-scale screening of isolates before performing their detailed

molecular investigation, therefore appropriate for use in developing countries.

As illustrated in Table 11, the results demonstrated that all strains can utilize

adonitol, L-arabinose, D-arabitol,D-cellobiose, D-fructose, L-fructose, D-galactose,

α-D-glucose, m-inositol, maltose, D-mannitol, D-mannose, L-rhamnose, D-sorbitol,

sucrose, D-trehalose, turanose, pyruvic acid methyl ester, L-alanine and glycyl-L-

glutamic acid, strengthening the fact that a wide array of carbon substrates are

utilized by fast-growing rhizobia (Van Rossum et al., 1995). These results are also

in line with the studies of Mohamed et al. (2000) and Arora et al. (2000) who stated

that fast-growing rhizobia utilize disaccharides most often as compared to slow-

growing rhizobia.Whereas all the isolated Rhizobium sp. were not able to utilize α-

cyclodextrin, i-erythritol, D-glucosaminic acid, α-keto butyric acid, L-

phenylalanine, thymidine, phenyethyl-amine and putrescine.

Antibiotic resistance is a widely used parameter for rhizobium classification

(Hungria et al., 2001), and the variability among isolates in terms of tested

antibiotics confirmed this test as a useful criterion for strain selection. The eight

isolated bacterial strains were tested for seven antibiotics and the result

isdemonstrated in Table 12. All tested isolates were resistant to chloramphenicol

and neomycine sulfate at 5 and 50 µg L-1, streptomycine sulfate and erythromycin

at 5, 50 and 100 µg L-1 and for ampicilin all strains were resistant at 5 µg L-1. In

contrast,all strains were susceptible to tetracycline hydrochloride and neomycine

108

sulfate at 100 and 300 µg L-1; chloramphenicol and ampicilin at 300 µg L-1. For

kanamycine

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Table 11: Carbom source utilization of Rhizobium strains isolated from groundnut nodules by using BIOLOG.

Carbon-source utilization BN-6 BN-12 BN-19 BN-20 BN-23 BN-25 BN-39 BN-50 α-Cyclodextrin - - - - - - - - Dextrin - + + + + + + + Glycogen - + w - - + + - Tween 40 - - - + - + - - Tween 80 - w - + - w w - N-acetyl-D-galactoseamine - + + + + + + + N-acetyl-D-glucoseamine - + + + + + + + Adonitol + + + + + + + + L-Arabinose + + + + + + + + D-Arabitol + + + + + + + + D-Cellobiose + + + + + + + + i-Erythritol - - - - - - - - D-Fructose + + + + + + + + L-Fructose + + + + + + + + D-Galactose + + + + + + + + Gentibiose - + + + + + + + α-D-Glucose + + + + + + + + m-Inositol + + + + + + + + α-D-lactose + + w + + w + + Lactulose - + - + + - + + Maltose + + + + + + + + D-Mannitol + + + + + + + + D-Mannose + + + + + + + + D-melibiose - + + + + - + + D-psicose - + + - + + + - D-raffinose - + + + + - + + L-Rhamnose + + + + + + + + D-Sorbitol + + + + + + + + Sucrose + + + + + + + +

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D-Trehalose + + + + + + + + Turanose + + + + + + + + Xylitol + + + - + - + + Pyruvic acid methyl ester + + + + + + + + Succinic acid mono-methyl-ester + + + - w - + + Acetic acid - + + + + + + + Cis-aconitic acid - + + + + - + + Formic acid - + w - + + + - D-galactonic acid - + - - + + + - D-galacturonic acid + - - - - - - - D-gluconic acid + + + - + + + - D-Glucosaminic acid - - - - - - - - D-glucuronic acid + + - - + + + + α-hydroxybutyric acid - + + - + + + + β-hydroxybutyric acid + - - - - - - - p-hydroxyphenylacetic acid + + - - - - - - Itaconic acid + - - - - - - - α-keto butyric acid - - - - - - - - α-keto glutaric acid - + + - w + + - α-keto valeric acid - + - - - - - - D,L-Lactic acid - + + + + + + + Malanoic acid - + - - - - - - Propionic acid - + + - + + + + Quinic acid - + + + + + + + D-saccharic acid + + - - - - - - Sebacic acid - + - - - - - - Succinic acid - + + + + + + + Bromosuccinic acid - + + + + + + + Succinamic acid - + w - + - w + Glucuronamide - + - - - - - - L-alaninamide - + - - + + + + D-alanine - - + - + - + +

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L-alanine + + + + + + + + L-alanyl-glycine - + + - + + + + L-asparagine - - + + + + + + L-aspartic acid - + + - + - + + Glycyl-L-glutamic acid + + + + + + + + L-glutamic acid - + + + + + + - L-histidine + + + - + + + + Hydroxy-L-proline - + + + + + + + L-ornithine - - + - + - + + L-phenylalanine - - - - - - - - L-proline - - + + + + + + L-pyroglutamic acid - + - - + + + + L-serine + + + - + + + + L-threonine w - w - + + + + D,L-carnitine + + - - - - - - γ-amino butyric acid + + w - + + + + Urocanic acid + + - - w + + + Uridine + + + - + + + + Thymidine - - - - - - - - Phenyethyl-amine - - - - - - - - Putrescine - - - - - - - - 2-aminoethanol - + w - w + + - 2,3-butanediol - w - - - + + - D,L-α-glycerol phosphate w - - - - - - - D-glucose-6-phosphate + + - - + - + +

+ Positive; - negative; w weakly utilized

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Table 12: Antibiotic resistant test of isolated Rhizobium strains.

+ Positive; - negative

Antibiotics ( µg ml-1) BN-6 BN-12 BN-19 BN-20 BN-23 BN-25 BN-39 BN-50 Tetracycline hydrochloride (5) + - + - - - - + Tetracycline hydrochloride (50) - - + - - - - - Chloramphenicol (100) + + + + - + + + Streptomycin sulfate (300) + + + - + + + + Ampicilin (50) + + + - - - + + Ampicilin (100) - - + - - - - - Neomycin sulfate (50) + + + + + + + + Erythromycin (300) - - + - - - - - Kanamycin sulfate (100) - - + - - - - - Kanamycin sulfate (300) - - + - - - - -

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sulfate only the strain BN-19 is resistant to all four tested concentrations (5, 50, 100

and 300 µg L-1), whereas other 7 isolates were susceptible.

4.4 BIOCHEMICAL CHARACTERIZATION OF EFFECTIVE PGPR

Seven potential PGPRs were selected on the basis of plant growth

promoting characterization to be evaluated in controlled and field environment.

Among these potential PGPRs, BN-2, BN-5, BN-44 and BN-55 were isolated from

groundnut nodules and GS-2, GS-4 and GS-6 were isolated from rhizospheric soil.

To carry out the biochemical characterization of these potential PGPRs API-20E

and API-ZYM kits were used.

API-20E kits were used to identify Gram-negative microorganisms. The kit

employs 20 biochemical tests which evaluated the ability of the microorganisms to

utilize different carbohydrates and amino acids sources. The biochemical results of

API-20E kits are illustrated in Table 13. The results depicted that all tested PGPRs

were positive for B-galactosidase and citrate utilization, whereas none was able to

synthesize H2S and inositol. Only the strain GS-4 was able to utilize all three amino

acids i.e., arginine, lysine and ornithine, however, BN-44 was positive for lycine

and BN-55 was positive for ornithine also. Indole was produced only byGS-4, and

saccharose, melibiose and amygdalin were utilized by GS-2, GS-4 and GS-6. All

strains proved to be negative for glucose and mannitol production except GS-4 and

GS-6, similarly sorbitol and rhamnose were consumed by BN-55, GS-2 and GS-4

only. For urease production, the strains BN-2, BN-5 and GS-4 were positive and

the strains BN-2, BN-44, BN-55 and GS-4 were positive for tryptophane

deaminase. All strains were negative for acetone production except BN-2 and BN-

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Table 13: Biochemical characterization of potential PGPRs used in pot experiment by using API 20E kits.

+ Positive, - negative

Characterization BN-2 BN-5 BN-44 BN-55 GS-2 GS-4 GS-6

B-galactosidase (ONPG) + + + + + + + Arginine dihydrolase (ADH) - - - ˗ - + - Lysine decarboxylase (LDC) - - + - - + - Orinthine decarboxylase(ODC) - - - + - + -

Citrate utilization (CIT) + + + + + + + H2S production - - - - - - - Urease + + - - - + - Tryptophane deaminase (TDA) + - + + - + - Indole production (IND) - - - - - + - Acetoin production (VP) + - - + - - - Gelatinase (GEL) - + - + - + + Glucose (GLU)

Sug

ars

ass

imila

tion

- - - - - + + Mannitol (MAN) - - - - + + + Inositol (INO) - - - - - - - Sorbitol (SOR) - - - + + + - Rhamnose (RHA) - - - + + - - Saccharose (SAC) - - - - + + + Melibiose (MEL) - - - - + + + Amygdalin (AMY) - - - - + + + Arabinose (ARA) - - + + + - +

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55. The strains BN-2, BN-44 and GS-2 were not able to utilize gelatinase, similarly

arabinose was not synthesized by BN-2, BN-5 and GS-4.

The API ZYM test offers a simple and reliable protocol for the

differentiation of different bacterial genus and species. The kit implies the

quantitative method formerly developed for the detection of enzymatic activity in

microorganisms by providing the systematic and prompt analysis of enzymatic

reactions by using very small amount of sample. The kit permits the synchronized

detection of 19 different enzymatic activities. The results of API ZYM kits for

experimental PGPRs are summarized in Table 14. The results examined that all

tested strains were able to synthesize enzymes alkaline phosphate esterase, esterase,

esterase lipase, leucine arylamidase, α-chymotrypsin, acid phosphatase and

naphthol-AS-BI-phosphohydrolase; whereas none of the tested isolates were able

to synthesize the enzymes lipase and α-fucosidase. All strains were negative for N-

acetyl-β-glucosidase except GS-4, similarly α-mannosidase was synthesized by

BN-55 only. For vanile arylamidase and cystine arylamidase only BN-2 and BN-44

were negative Trypsin was produced by all strains except BN-5, BN-44 and GS-6

and α-galactosidase was synthesized by BN-2, BN-44 and GS-4 only.

On the basis of plant growth promoting characteristics (P-solubilization,

IAA production, nifH gene amplification, catalase and oxidase production) and

biochemical characterization, seven isolated bacterial strains were screened to be

tested in pot and field experiments (Table 15). Three Rhizobium sp. (BN-19, BN-20

and BN-30) were also inoculated in pot and field as indigenous Rhizobiumsp. to

nodulate groundnut.

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Table 14: Biochemical characterization of potential PGPRs used in pot experiment by using API ZYM kits.

Characteristics BN-2 BN-5 BN-44 BN-55 GS-2 GS-4 GS-6 Alkaline Phospahate Estarase(C4) + + + + + + + Esterase (C4) + + + + + + + Estarase Lipase + + + + + + + Lipase(C14) - - - - - - - Leucine arylamidase + + + + + + + Vanile arylamidase - + - + + + + Cystine arylamidase + + - + + + + Trypsin + - - + + + - α-Chymotrypsin + + + + + + + Acid phosphatase + + + + + + + Naphthol-AS-BI-phosphohydrolase + + + + + + + α-galactosidase - + - + + - + N-acetyl-β-glucosidase - - - - - + - α-mannosidase - - - + - - - α-fucosidase - - - - - - -

+ Positive; - negative

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Table 15: PGP characteristics of bacterial strains used in pot and field experiment

Strain ID

Accession number

P-solubilization

IAA Production

Oxidase Catalase nifH gene

Highest Similarity (%)

BN-2 AB971370 362 ± 4 41 ± 2 - + - Bacillus safensis BN-5 AB971373 432 ± 1 80 ± 2 + + - Arthrobacter globiformis BN-44 AB982441 674 ± 8 45 ± 2 - + - Pseudomonas oryzihabitans BN-55 AB982446 611 ± 3 96 ± 3 - + - Pseudomonas psychrotolerans GS-2 AB773830 301 ± 4 58 ± 1 + + - Kocuria turfanensis GS-4 AB773832 562 ± 5 66 ± 1 - + - Bacillus aryabhattai GS-6 AB773834 450 ± 1 98 ± 4 + + - Microbacterium arborescens BN-19 AB854065 152 ± 1 11 ± 2 + + + Rhizobium alkalisoli BN-20 AB969784 56 ± 3 66 ± 2 + + + Rhizobium huautlense BN-23 AB969785 290 ± 4 49 ± 2 - + + Rhizobium pusense

+ Positive; - negative Burkholderia ferrariae FeGl01 and Burkholderia cepacia ATCC 25416 are a standard reference strains for P-solubilization having the potential to solubilize inorganic P in the range of 97.7 and 88.2 µg mL-1,respectively (Inui-Kishiet al., 2012). Azotobacter vinelandii Mac 259 and Bacillus cereus UW 85 are a standard reference strains for IAA production having the potential to synthesize IAA in the range of 20.9 and 40.2 µg mL-1,respectively. (Paterno, 1997).

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4.5 INOCULATION EFFECT OF PGPR AND Rhizobium sp. IN POT

EXPERIMENT

In order to evaluate the proficiency of selected PGPRs and Rhizobium sp.

on growth parameters and N2-fixation of groundnut, pot experiment was conducted

under controlled green house environment. Selected seven PGPRs (BN-2, BN-5,

BN-44, BN-55, GS-2, GS-4 and GS-6) were applied individually in each

replication along with consortium of three Rhizobium sp. (BN-19, BN-20 and BN-

23). Crop data was collected to determine the impact of treatments on groundnut

yield and nitrogen fixation as compared to control.

4.5.1 Inoculation Effect on Growth Parameters of Groundnut in Controlled

Condition

The data illustrating the impact of co-inoculation of PGPR and Rhizobium

sp. on nodule number, pod and biomass yield of groundnut is presented in Table

16. The results revealed that all the treatments have significant impact on nodule

number per plant over control, but maximum nodule (47 per plant) was recorded in

treatment T7 (GS-4; Bacillus aryabhattai), followed by T4 (BN-44;

Pseudomonasoryzihabitans) and T5 (BN-55; Pseudomonas psychrotolerans)

having nodulenumber 44 per plant each, exhibiting the increase of 104% and 91%

over control respectively. Minimum nodule number (28 per plant) was observed in

treatment T6 (GS-2; Kocuria turfanensis) having only 22% increase over control.

These results are similar to the findings of Bai et al. (2003) who concluded an

increase in nodule number of soybean crop when the plant was coinoculated with

Bradyrhizobium japonicum and Bacillus sp.Badawi et al. (2011) reported an

increase in nodule number and nodule dry weight by 61% over control when

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Table 16: Co-inoculation of PGPR and Rhizobium sp. illustrated a significant effect on growth parameters of groundnut

under pot experiment.

Treatments Nodule

No Pod

Yield Biomass

Yield no plant-1 g plant-1 T1 Control 23 d 10 e 16 e T2 BN-2; Bacillus safensis 31 bc 11 de 19 de T3 BN-5; Arthrobacter globiformis 36 b 13 d 21 cd T4 BN-44; Pseudomonas oryzihabitans 44 a 16 bc 23 bc T5 BN-55; Pseudomonas psychrotolerans 44 a 19 b 25 b T6 GS-2; Kocuria turfanensis 28 cd 16 c 26 b T7 GS-4; Bacillus aryabhattai 47 a 22 a 32 a T8 GS-6; Microbacterium arborescens 33 bc 24 a 34 a

LSD 6.1805 2.6908 3.5155

Rhizobium sp. BN-19, BN-20 and BN-23 are added in all treatments except control.

The mean (n=3) with different small letters indicate significant difference at probability level < 0.05.

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groundnut was coinoculated with Bradyrhizobiun sp. and PGPR. Increase in

nodulation and nitrogen fixation of legumes by co-inoculation with PGPR is

becoming a useful mean to enhance N2 availability in agricultural production

system (Abdel-Wahab et al., 2008). The mechanism of promoting nodulation and

nitrogen fixation of legumes by PGPR could be the production of flavonoid like

compounds, auxins and siderophores which can promote nodulation by stimulating

the host legume to produce more flavonoid signal molecules and also the root

growth stimulation by phytohormones synthesized by PGPR aided in increased

sites for bacterial infection and nodulation (Bai et al., 2002; Vessey and Buss,

2002). Kloepper (2003) and Verma et al. (2010) also reported enhanced nodulation

by PGPRs as well as formation of more infection sites on the hairs and epidermis

of the roots of leguminous plant.

These results indicated that the occurrence of indigenous rhizobia of

groundnut in the experimental soil is not sufficient, therefore resulted in reduced

nitrogen fixation efficiency in control. This finding revealed that the use of

effective groundnut rhizobia is requisite for maximum nitrogen fixed by the crop.

These results were also supported by El-Sawy et al., 2006 and Mekhemar et al.,

2007. Groundnut inoculation with rhizobia improved nodulation, resulting in

significant increase in number and dry weight of nodules as compared to the

uninoculated control. This can be noted from the remarkable differences between

the inoculated and uninoculated treatments and accentuated the need to inoculate

groundnut seeds with their effective rhizobial strains (Vlassak and Vanderleyden,

1997).

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The data illustrating the effect of co-inoculation of PGPRand Rhizobium sp.

on pod yield of groundnut is presented in Table 16. The results elicited the

significant increase in pod yield by application of PGPR along with Rhizobium as

compared to uninoculated treatment (T1). Maximum pod yield (24 per plant) was

observed in treatment T8 (GS-6; Microbacterium arborescens) with 140% increase

over control (T1). Treatments T7 (GS-4; Bacillus aryabhattai) and T5 (BN-55;

Pseudomonas psychrotolerans) exhibited 22 and 19 pods per plant respectively,

having percent increase of 120% and 90% over control. Groundnut-affiliated

bacteria isolated from diverse environment like rhizospheric soil, phylloplane and

seed endophytes prompted the early plant growth in the greenhouse. These results

are similar to the findings of Dey et al. (2004), who affirmed 32% increase in pod

yield of groundnut over control when inoculated with rhizobacteria. Asghar et al.

(2002) also stated an increase in plant height, pod and grain yield and oil contents

of groundnut when seeds were inoculated with PGPRs. Similarly, 37% increase in

soybean pod yield over control was observed by seed inoculation with PGPR and

effective Rhizobium strains (Dileepkumar and Dube, 1992). PGPRs have proved to

enhance the yield and quality of many legumes, when they are coinoculated with

rhizobia. This synergetic effect can be attributed to their ability to increase the N2-

fixation, along with their potential to improve nutrients availability and uptake

from soil, resulting from the synthesis of hormones and siderophores, phosphate

solubilization and improvement of water and nutrients uptake. Solubilization of

insoluble phosphate by PGPR resulted in increased availability of P to the plants;

hence elevating growth characteristics by improving N and P uptake in plants,

ultimately enhancing yield and yield components. The same results are supported

by Yadav and Verma (2009) and Verma et al. (2010).

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A considerable impact was observed on biomass yield of groundnut when

the inoculum is applied as compared to control (T1). As it is inferred from the data

presented in Table 16, that maximum biomass yield (34 g plant-1) was obtained

from the treatment T8 (GS-6; Microbacterium arborescens) which is 113% greater

as compared to control. Treatment T7 (GS-4; Bacillus aryabhattai) yielded 32 g

plant-1, producing 100% more biomass than control. The minimum yield was

observed in treatment T2 (BN-2; Bacillus safensis) where the increase in yield as

compared to control was only 19%, however there was a consistent increase in

biomass from treatments T3 to T6, exhibiting the biomass increase from 21 to 26 g

plant-1. The increase in biomass by PGPR application is also observed in other

legumes like chickpea (Trapero-Casas et al., 1990) and soybean (Zhang et al.,

1997), and it has been revealed that microbes which enhance early plant

development are prompt colonizers of rhizosphere.Similarly 26% increase in

biomass yield was obtained by application of phylloplane bacterial isolate Bacillus

megaterium, Pseudomonas aeruginosa and Bacillus firmis on groundnut (Kishore

et al., 2005).

These effective results of inoculation could be attributed to the beneficial

effects of PGPR, resulting in improved root growth, thus aiding in nutrient uptake

(Zahir et al., 2004; Abdel-Wahab et al., 2008). The beneficial impacts of PGPRs

could enhance plant growth and the metabolism of photosynthates thereby

improving plant biomass (Tilak et al. 2005; Verma et al. 2010). This increase in

plant biomass may be ascribed to the release of root exudates by PGPRs including

plant growth promoting hormones like indole acetic acid, gibberellins and

cytokinines (Badawi et al., 2011).

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4.5.1 Inoculation Effect on N2-Fixation of Groundnut in Controlled

Condition

There is consistent variation in total shoot nitrogen contents among all the

treatments (Table 17). Maximum shoot N contents (1.5%) were recorded in

treatments T5 (BN-55; Pseudomonas psychrotolerans), T7 (GS-4; Bacillus

aryabhattai) and T8 (GS-6; Microbacterium arborescens) and minimum (1.4%)

were recorded in treatments T2 (BN-2; Bacillus safensis), T3 (BN-5; Arthrobacter

globiformis), T4 (BN-44; Pseudomonas oryzihabitans) and T6 (GS-2; Kocuria

turfanensis). The total shoot N contents of 1.3% were recorded in uninoculated

treatment (control T1).

The coinoculation of Rhizobium sp. with PGPR strains has a significant

effect on the amount of nitrogen fixed by groundnut (Table 17). Treatments T5

(BN-55; Pseudomonas psychrotolerans), T7 (GS-4; Bacillus aryabhattai) and T8

(GS-6; Microbacterium arborescens) resulted in maximum amount of nitrogen

fixed (15 mg g-1), followed by treatment T6 (GS-2; Kocuria turfanensis) which

fixed nitrogen in the range of 11 mg g-1, indicating that P. psychrotolerans, M.

arborescens and B. aryabhattaiproved to be the promising PGPR for enhancing the

symbiotic activity of Rhizobium sp. The percent increase in N2-fixed by T5, T7 and

T8 is 150% as compared to T1 (control) which fixed only 6 mg g-1 of nitrogen.

Minimum N2 (8 mg g-1) was fixed by treatment T2 (BN-2; Bacillus safensis) with

percent increase of only 33.3% over control.

The proportion of nitrogen derived from atmosphere (%Ndfa) exhibited the

same pattern as followed by the quantity of nitrogen fixed (Table 17). Co-

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Table 17: Effect of co-inoculation of PGPR and Rhizobium sp. on N2-fixation of groundnut in pot experiment.A

significant increase was recored in N2-fixation by Rhizobium application owing to their potential of fixing atmospheric

nitrogen.

Treatments Shoot N Ndfa Shoot N

uptake N2-fixed

----------%---------- ----------mg g-1--------- T1 Control 1.3 b 31 e 13 b 6 e T2 BN-2; Bacillus safensis 1.4 b 38 d 14 b 8 d T3 BN-5; Arthrobacter globiformis 1.4 b 42 d 14 b 9 d T4 BN-44; Pseudomonas orizihabitans 1.4 b 49 c 14 b 10 c T5 BN-55; Pseudomonas psychrotolerans 1.5 a 68 a 15 a 15 a T6 GS-2; Kocuria turfanensis 1.4 b 56 b 14 b 11 b T7 GS-4; Bacillus aryabhattai 1.5 a 70 a 15 a 15 a T8 GS-6; Microbacterium arborescens 1.5 a 70 a 15 a 15 a

LSD 0.0562 4.8264 0.5620 0.0580



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inoculation of PGPR with Rhizobium sp. enhanced the share of nitrogen which

plants acquired from biological fixation as compared to uninoculated treatment

(control). Plants inoculated with the strains GS-4; Bacillus aryabhattai and

Rhizobium sp. (T7) and GS-6; Microbacterium arborescens and Rhizobium sp. (T8)

received 70% of their N2 demand from atmosphere, which exhibited a 126%

increase over control. This was followed by treatment T5 (BN-55; Pseudomonas

psychrotolerans) which fixed 68% nitrogen from atmosphere, resulted in 119%

increase over control.

Our results are in correspondence with previous studies demonstrating the

growth promoting activities of PGPRs identified as Pseudomonas spp.

andAzospirillum spp. on rhizobium-legume symbiotic effectiveness (Bashan et

al.,2004; Valverde et al., 2006). The combined inoculation of groundnut seed

withRhizobium sp. and PGPR lead to enhanced production of nodules interpreted

by greater shoot N accumulation and improved seed yield (Figueiredo et al., 2007).

Dubey (1996) also stated that coinoculation of Bradyrhizobium sp. and P.

striata enhanced biological nitrogen fixation in soybean. Studies also suggested that

combined inoculation of Bradyrhizobium with P. fluorescens improved the nodule

occupancy in soybean (Fuhrmann and Wollum, 1989).

The nitrogen fixation in beans was enhanced by inoculation of PGPRalong

with Rhizobium (Yadegari et al., 2010), resulted in increase of average value of

%Ndfa to 42 and 49% as compared to control. This difference in N2-fixation and %

Ndfa is probably attributed to siderophore production and ability of P. fluorescens

for auxin synthesis and P-solubilization. As siderophores performed an

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appreciablerole in increasing nodule dry weight and therefore enhancing biological

nitrogen fixation because the nitrogenase enzyme needed a lot of iron (Fe) (Catellan

et al., 1999). Biological nitrogen fixation also demanded a good amount of the

absorbed phosphate from soil to synthesize ATP which is requisite for atmospheric

nitrogen fixation through groundnut–rhizobia symbiosis. Therefore, constant

availability of phosphorous is essential to sustain the process of biological nitrogen

fixation, which could be possible by the process of phosphate solubilization by

PSMs.

4.6 INTEGRATED EFFECT OF PGPR AND CHEMICAL

FERTILIZERS ON GROUNDUT YIELD AND N2-FIXATION IN

FIELD CONDITION

On the basis of performance of potential PGPR in green house experiment

and by considering the percent increase of different treatments over control (Table

18), three potential bacterial strains BN-55(Pseudomonas psychrotolerans),GS-4

(Bacillus aryabhattai)and GS-6 (Microbacterium arborescens) were selected to be

used in consortium under field conditions along with Rhizobium species BN-19 (R.

alkalisoli), BN-20 (R. huautlense) and BN-23(R. pusense). PGPR and Rhizobium

sp. were applied along with full (20 and 80 kg ha-1) and half (10 and 40 kg ha-1)

recommended doses of chemical fertilizers i.e. N and P. Crop data was obtained to

assess the effect of treatments on crop yield parameters including biomass and pod

yield and nitrogen fixation as compared to control.

4.6.1 Integrated Effect of Co-inoculation of PGPR and Chemical Fertilizers

on Growth Parameters of Groundnut

127

128

Table 18: Percent increase in yield and N2-fixation of groundnut over control

under pot experiment.

Treatments Nodule

No Pod Yield Biomass

Yield Ndfa Shoot N

uptake N2-fixed

T2 BN-2 34.3 13.8 16.3 20.8 0.5 21.5 T3 BN-5 52.9 31.0 28.6 32.7 3.0 36.6 T4 BN-44 88.6 69.0 38.8 56.7 3.5 62.2 T5 BN-55 88.6 96.6 53.1 118.4 8.7 137.4 T6 GS-2 18.6 65.5 42.9 79.6 0.5 80.6 T7 GS-4 102.9 131.0 98.0 121.8 8.2 140.1 T8 GS-6 42.9 148.3 108.2 121.8 8.4 140.8

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The data on the growth parameters of groundnut as affected by co-

inoculation of PGPR and Rhizobium sp. along with full and half recommended

doses of chemical fertilizers is presented in Table 19. The data regarding the plant

density of groundnut illustrated that there is no significant effect on crop density by

application of chemical fertilizers alone and also by combined application of

PGPRs and chemical fertilizers. Maximum plant density (12 plants m-2) was

observed in treatment T3 (NP @ 10 & 40 kg ha-1) exhibiting only 12% increase

over control. The main reason for low and non-significant plant density was thelow

moisture availability during the sowing time which resulted in poor germination.

There was no rainfall in experimental site for almost one month after sowing,

leading an adverse impact on germination and seedling emergence.

As demonstrated in Table 19, the data of biomass yield indicated a

significant difference among the treatments. Maximum biomass yield (9.2 t ha-

1)was observed in treatment T4 (BN-55 + GS-4 + GS-6 + NP @ 20 & 80 kg ha-1) in

which co-inoculation of PGPR and Rhizobium sp. was applied with full

recommended dose of chemical N and P, exhibiting 80% increase over control

(T1). This increase in biomass yield was followed by T2 (NP @ 20 & 80 kg ha-1)

and T5 (BN-55 + GS-4 + GS-6 + NP @ 10 & 40 kg ha-1) having 67 and 45%

increase in yield over control, thereby expressing a consistent increase in biomass

yield among the treatments.

The present study confirmed the results of Latif et al. (2009) and Kishore et

al. (2005) who reported 40 and 26% increase in biomass yield over control by

application of phylloplane, endophytic and rhizosphere bacteria along with

chemical fertilizers in groundnut. Similarly, Heip et al. (2002) and Lanier et al.

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Table 19: Effect ofco-inoculation of PGPR and Rhizobium sp. with and without NP fertilizer on groundnut yield under

field condition.A significant increase in yield was recorded due to the ability of PGPR to increase nutrients avialibity

to the plants.

Treatments Plant

density Biomass

yield Pod yield

m-2 --------t ha-1-------- T1 Control 10.7 5.1 f 2.1 e T2 NP @ 20 & 80 kg ha-1 11.7 8.5 b 3.2 b T3 NP @ 10 & 40 kg ha-1 12 6.3 e 2.6 cd T4 BN-55 + GS-4 + GS-6 + NP @ 20 & 80 kg ha-1 11.3 9.2 a 3.7 a T5 BN-55 + GS-4 + GS-6 + NP @ 10 & 40 kg ha-1 11 7.4 c 3.1 bc T6 BN-55 + GS-4 + GS-6 11.7 7.0 d 2.3 de LSD 0.3954 0.4821



PGPRs: (BN-55: Pseudomonas psychrotolerans + GS4: Bacillus aryabhattai + GS-6: Microbacterium arborescens)

131

(2005) reported an increase in groundnut biomass yield as compared to control

when bacterial inoculum was applied. Co-inoculation of the common bean with

Rhizobium sp.andPseudomonas fluorescens enhanced shoot dry weight and

biomass yield (Yadegari et al., 2010). Co-inoculation of Azospirillum and

Pseudomonas significantly increased various plant growth parameters like plant

biomass, shoot N contents, leaf size, plant height and root length in cereal crops,

therefore they have the potential to be used as natural fertilizers (Bashan et al.,

2004). It has been believed that the synthesis of bacterial auxin leads to the

expansion of lateral roots, resulting in increased root surface area enabling the plant

to assimilate more nutrients and water from soil, hence increasing root and plant

biomass (Vessey and Buss, 2002; Taiz and Zeiger, 2010). Rahman et al. (1992)

concluded significant increase in plant biomass yield when bacterial inoculum was

applied in combination with phosphatic fertilizers. Similarly, Ramesh and Sabale

(2001) observed that seed inoculation with P solubilizer bacteria along with

application of 33 kg ha-1 phosphatic fertilizer significantly increased dry matter and

yield of groundnut as compared to other treatments.

The results of pod yield of groundnut as affected by consortium of PGPR

and Rhizobium sp. along with full and half recommended doses of N and P

fertilizers are illustrated in Table 19. The trend of increase in pod yield among

treatments is same as that of biomass yield. Maximum pod yield (3.7 t ha-1) is

obtained from treatment T4 (BN-55 + GS-4 + GS-6 + NP @ 20 & 80 kg ha-1),

followed by T2 (NP @ 20 & 80 kg ha-1) and T5 (BN-55 + GS-4 + GS-6 + NP @ 10

& 40 kg ha-1) exhibiting 3.2 and 3.1 t ha-1 pod yield respectively. The maximum

percent increase in pod yield was 76% between treatment T4 and T1 (control).

132

These findings are in line with those of Latif et al. (2009) who documented 33%

increase in groundnut pod yield as compared to control, when bacterium inoculum

was applied along with N and P fertilizers @ rate of 20 and 80 kg ha-1. Similarly,

Mehta and Rao (1996) revealed that inoculation of groundnut with Rhizobium sp. at

NP level of 15 and 44 kg ha-1 significantly increased pod yield. It was also

confirmed that P application rates from 40-120 kg ha-1 increased groundnut crop

yield from 17-54% (Mandimba and Djondo, 1996). Musa et al. (2003) reported an

increase of 44% in pod yield of groundnut by the combined inoculation of

Bradyrhizobium along with Diazotroph @ 10 and 20 kg ha-1 N fertilizer, however

N application at the rate of 40 kg ha-1 decreased nodulation and N2-fixation.

Raychaudhuri et al. (2003) recorded maximum pod yield of 1740 kg ha-1 with the

dual inoculation of Rhizobium and phosphate solubilizing microorganisms. In

another study the combined application of chemical fertilizer and bacterial

inoculum significantly increased pod yield over control (Kishore et al., 2005).

The major reason for increased biomass and pod yield with 20 and 80 kg

ha-1 N and P treatments might be due to the fact that application of P fertilizer

stimulates better root development, hence maximizing nutrients and moisture

absorbing area in the soil system (Latif et al., 2009). In addition, the efficient root

system also complements the activity of N2-fixing bacteria. The positive role of

phosphatic fertilizer in improving legumes yield is reported by several researchers

(Ahad et al., 2002; García et al., 2004). The combined use of inorganic fertilizers

and bacterial inoculation could increase the instinctive nutrients supplying ability

of the soil for both macro and micronutrients, and also improve soil physical

properties resulting in extensive rooting, enhanced nutrients uptake by the plants,

133

increase in leaf area, dry matter production and grain yield (Shoghi-Kalkhoran et

al., 2013). The integrated fertilization system provides more nitrogen to the crops

and increase protein contents, in addition to reducing nitrogen loss. This might be

due to the presence of N2-fixing bacteria which provide biologically fixed N2 to the

plants along with secretion of beneficial plant growth promoting substances like

IAA, gibberellins, riboflavin and siderophores (Hayat et al., 2010).

4.6.2 Integrated Effect of Co-inoculation of PGPR and Chemical Fertilizers

on N2-Fixation of Groundnut

Efficacy of nitrogen fixation ability is indicated by accumulation of N2 in

plant tissues. Data in Table 20 illustrated that there is a consistent increase in shoot

N concentration among all treatments. Maximum shoot N contents (1.4%) was

observed in treatments T2 (NP @ 20 & 80 kg ha-1), T3 (NP @ 10 & 40 kg ha-1), T4

(BN-55 + GS-4 + GS-6 + NP @ 20 & 80 kg ha-1) and T6 (BN-55 + GS-4 + GS-6)

with only 7.7% increase over control. Shoot N uptake data (Table 20) was 67.5 kg

ha-1 in uninoculated treatment (T1) and magnified to 127.9 kg ha-1 in treatment T4

(BN-55 + GS-4 + GS-6 + NP @ 20 & 80 kg ha-1) as a result of coinoculation of

PGPR and Rhizobium sp. along with recommended doses of NP fertilizers. The

percentage increase in shoot N uptake was 90% as compared to control. This

increase was followed by treatments T2 (NP @ 20 & 80 kg ha-1) and T5 (BN-55 +

GS-4 + GS-6 + NP @ 10 & 40 kg ha-1), resulting in 71 and 45% increase over

control respectively. These results proved that co-inoculation of groundnut with

PGPR and Rhizobium sp. followed by inorganic fertilizer treatment resulted in

obvious accumulation of nitrogen in plant shoot tissues. Therefore, our results

highlighted the key role of PGPR co-inoculation in the advancement of biological

134

Table 20: Effect of co- inoculation of effective PGPR and Rhizobium sp. with and without NP fertilizer on N2-fixation

of groundnut under field experiment.The significant increase in N2-fixation as compared to control is attributed to the

potential of Rhizobium sp. to fix atmospheric nitrogen.

Treatments Shoot N

content Ndfa Shoot N

uptake N2-fixed

---------%--------- ------kg ha-1------ T1 Control 1.3 b 36.0 e 67.5 e 36.9 e T2 NP @ 20 & 80 kg ha-1 1.4 a 38.9 e 115.5 b 67.4 c T3 NP @ 10 & 40 kg ha-1 1.4 a 44.0 d 87.6 d 57.9 d T4 BN-55 + GS-4 + GS-6 + NP @ 20 & 80 kg ha-1 1.4 a 56.3 b 127.9 a 108.0 a T5 BN-55 + GS-4 + GS-6 + NP @ 10 & 40 kg ha-1 1.3 b 68.4 a 97.6 c 98.2 b T6 BN-55 + GS-4 + GS-6 1.4 a 49.1 c 94.3 cd 69.4 c LSD 0.0563 4.9815 7.8142 8.4433



PGPRs: (BN-55: Pseudomonas psychrotolerans + GS4: Bacillus aryabhattai + GS-6: Microbacterium arborescens)

135

nitrogen fixation by groundnut-rhizobia system and aided in maximizing the root

biomass and thus improved absorption of nutrients from the surroundings. These

results are also supported by the findings of Abdel-Wahab et al. (2008) and Verma

et al. (2010). Hafner et al. (1992) also concluded that the concentration of shoot N

at mid pod filling stage was higher by integrated application of P and bacterial

inoculums as compared to treatment where only P fertilizer was applied, and the

total N uptake improved by 104%.

There was a significant effect on the quantity of nitrogen fixed by co-

inoculation of Rhizobium with PGPR (Table 20). Maximum N (108 kg ha-1) was

fixed by treatment T4 (BN-55 + GS-4 + GS-6 + NP @ 20 & 80 kg ha-1) and

thepercent increase over control was 193%. This increase in N-fixed was followed

by treatments T5 (BN-55 + GS-4 + GS-6 + NP @ 10 & 40 kg ha-1) andT6 (BN-55

+ GS-4 + GS-6) representing 166 and 88% increase over control (T1).These results

indicated that inoculating the groundnut plants with Rhizobium sp. resulted in a

significant increase in total N fixation as compared to control which dependent

only on indigenous soil Rhizobium sp. Similar results were concluded by Boddey et

al. (1990) who stated an increase in groundnut N contents by atmospheric N2-

fixation after inoculation with Bradyrhizobium sp. Increase in nitrogen fixation by

inoculation and fertilizer application was also reported by Herridge and Rose

(2002) and Boahen et al. (2002). The favorable response especially at phosphorous

fertilizers levels might be due to better root development by theapplication of P as

the prolific root system also enhances the activity of N-fixing bacteria (Latif et al.,

2009). Heip et al. (2002) revealed that in groundnut, theoptimum fertilizer mixture

and innoculum resulted in greatest economic results. From the present results it

136

could be suggested that there was a synergetic effect of nitrogen fixation, as the

inoculated treatments have both fixed more N and derived more N from the soil.

The percentage of N obtained from atmosphere (%Ndfa) are similar to the

results of N-fixed (Table 20), exhibiting a significant effect of PGPR and

Rhizobium inoculation along with chemical fertilizers on %Ndfa. Maximum nitrogen

from the atmosphere (68.4%) was derived by treatment T5 (BN-55 + GS-4 + GS-6

+ NP @ 10 & 40 kg ha-1) and the percent increase over control was 90%. Plants

inoculated with treatments T4 (BN-55 + GS-4 + GS-6 + NP @ 20 & 80 kg ha-1)

and T6 (BN-55 + GS-4 + GS-6) received 56 and 49% of their nitrogen requirement

from atmosphere, indicating 56 and 37% increase over uninoculated treatment (T1),

respectively. The reason for high %Ndfa by treatments T4 and T5 may be attributed

to the fact that P fertilizer enhances nitrogen fixation through its effect on

nodulation and thereby derives more nitrogen from the atmosphere (Lekberg and

Koide, 2005). The results of our study are in accordance with those of Cadisch et

al. (2000) and Latif et al. (2009) who observed that the proportion of %Ndfa from N

fixation ranged between 45 and 54% by using the 15N natural abundance technique.

They further concluded that under conditions of relatively high plant available 15N

conditions in the soil, the natural abundance technique is a feasible method to

access the N2-fixation.

4.7 VALIDATION OF NOVEL SPECIES FROM GROUNDNUT

NODULES ASRhizobium pakistanensis BN-19T

Among the eight Rhizobium sp. isolated from groundnut nodules, one strain

(BN-19T) formed a distinct 16S rRNA gene type by showing less than 97% 16S

137

rRNA gene similarity with its closely related species. Therefore this strain was

characterized for its taxonomic status by using phlylogenetic investigations

including housekeeping genes (atpD, recA and glnII) sequences, cellular fatty acid

profiles, DNA-DNA hybridization and phenotypic characterization.

For the validation of BN-19T, polyphasic taxonomic experiments were

performed in Rhizobium Research Center, China Agricultural University, Beijing,

China. Five reference strains of the genus Rhizobium were used namelyR. alkalisoli

CCBAU 01393T, R. tarimense PL-41T, R. vignae CCBAU 05176T, R. huautlense

SO2T, and R. leguminosarum USDA 2370T for DNA-DNA relatedness, fatty acid

analysis, physiological and biochemical tests. These reference strains were

compared with BN-19T under the same laboratory conditions. Considering the

sequence analysis of 16S rRNA and housekeeping genes, the strain BN-19T was

phylogenetically related to the members of genus Rhizobium, but distinct from all

the defined species thus representing a novel species in the genus Rhizobium, for

which the name Rhizobium pakistanensis sp. nov. was proposed. The type strain is

BN-19T (=LMG 27895T=CCBAU 101086T) andis deposited in two international

culture collections i.e., Culture Collection of China Agricultural University

(CCBAU=101086) and Universiteit Gent-Laboratorium voor Microbiologie

(LMG=27895).

4.7.1 Morphology and Phenotypic Characterization

The type strain BN-19T formed circular, smooth, covex and white colonies after

two days of incubation in YMA medium at 28°C. Cells were gram negative,

aerobic and non-motile. Electron microscopic study revealed that the cells were rod

138

shaped, 0.4-1.03 µm long and 0.3-1.2 µm wide (Figure 25). The phenotypic

characterization of the strain BN-19T in comparison to the reference strains are

presented in Table 21. The results depicted that 69 features differentiate BN-19T

from its closely related strains, whereas 26 were common to all tested strains. BN-

19T can utilize dextrin, N-acetyl-D-glucoseamine, adonitol, L-arabinose, D-arabitol,

D-cellobiose, gentibiose, m-inositol, maltose, D-mannitol, D-mannose, D-

melibiose, D- fructose, D-galactose, D-mannose, L-rhamnose, L-asparagine, D-

sorbitol, sucrose, D-trehalose, turanose, xylitol, β-methyl-D-glucoside, α-D-

glucose, D-raffinose, acetic acid, propionic acid, quinic acid, succinic acid,

bromosuccinic acid, D-alanine, L-alanine, L-aspartic acid, D-gluconic acid, , L-

histidine, hydroxy-L-proline, L-leucine, L-ornithine, L-serine, inocine and

uridinebut cannot utilize i-erythritol, α-cyclodextrin, citric acid, cis-aconitic acid,

D-galactonic acid lactone, tween 40, tween 80, D-galacturonic acid, D-

glucosaminicacid, D-glucuronic acid, α-Keto butyric acid, α-Keto valeric acid, D-

saccharic acid, L-alaninamide, D-serine, thymidine, phenyethyl-amine, 2,3-

butanediol, glycerol, D,L-α-glycerol phosphate, α-D-Glucose-1-phosphate, D-

Glucose-6-phosphate, and lactulose. α-D-lactose, formic acid, α-hydroxybutyric

acid, L-threonine and γ-Amino butyric acid are utilized weakly.Growth occurs

between 20-37°C; pH 5-10 and YMA supplied with 0-3% NaCl, with optimum

growth at 28°C, pH 7.0 havingNaCl concentration of 0.5%. BN-19T was resistant to

tetracycline hydrochloride and neomycin sulfate at 5 and 50 µg ml-1;

chloramphenicol, ampicilin and kanamycin sulfate at 5, 50 and 100 µg ml-1 and for

streptomycin sulfate and erythromycin it was resistant to all four tested levels of

antibiotics. The comparison of antibiotic resistance for BN-19T and reference

strains is depicted in Table 21.

139

4.7.2 Phylogenetic Analysis of 16S rRNA and Housekeeping Genes

An almost complete 16S rRNA gene sequence (1358 nucleotides) of strain

Figure 25: Scanning electron microscopic picture of BN-19T exhibiting the rod

shaped structure of Rhizobium.

140

Table 21:Differential features for type strains of R. pakistanensis BN-19T sp. nov.

andrelated reference species.

Characteristics 1 2 3 4 5 6

Dextrin + + - - - + N-Acetyl-D-glucoseamine + + - w + + Adonitol + + - w + + L-Arabinose + + - + + + D-Arabitol + + - + + + D-Cellobiose + + - + + + i-Erythritol - - - - - + Gentibiose + + w - + w m-Inositol + + w + + - α-D-Lactose w + - w - + Maltose + + - + + + D-Mannitol + + - + + + D-Mannose + + - + + + D-Melibiose + w + - + - β-Methyl-D-glucoside + - + - - + D-Psicose + - + - w + D-Raffinose + - + - + + D-Trehalose + + w + + + Turanose + + - + + + Succinic acid mono-methyl-ester

+ - - + w w

Acetic acid + + + + + - Cis-aconitic acid + + w w + + Citric acid - - + - - - Formic acid w + w + + - D-Galactonic acid lactone - + + + - w D-Galacturonic acid - - + - + + D-Gluconic acid + + + + w - D-Glucosaminic acid - - + - - w D-Glucuronic acid - - + w + w α-Hydroxybutyric acid w + w - w - β- Hydroxybutyric acid + + - + + w α-Keto butyric acid - + + - w -

141

α-Keto valeric acid - - + - - - Malanoic acid - + w w + w Propionic acid + - + - + - Quinic acid + - + + + - D-Saccharic acid - - + w - - Succinic Acid + + - + + + Bromosuccinic acid + - - + + + Succinamic acid w - + + + + Glucuronamide - - + w + - L-Alaninamide - - + w - + D-Alanine + w + - - - L-Alanine + w + + + - L-Aspartic acid + - w + - w Glycyl-L-aspartic acid + + w + - - Glycyl-L-glutamic acid + + - + w w L-Histidine + + - + + w Hydroxy-L-proline + - w - + - L-Leucine + + w - + - L-Ornithine + + + w + - L-Phenylalanine - - + - - - L-Pyroglutamic acid - - + - - - D-Serine - - + - - - L-Serine + + + w + - L-Threonine w - + - + - D,L-Carnitine - - + w + + γ-Amino butyric acid w - - w - + Urocanic acid - w - + + w Inocine + - - - - - Uridine + + - + + + Thymidine - - w + - - Phenyethyl-amine - - w w - w 2-Aminoethanol w - + + - - 2,3-Butanediol - - + - - w Glycerol - + + + + + D,L-α-Glycerol phosphate - - + + - - α-D-Glucose-1-phosphate - + + w + - D-Glucose-6-phosphate - + w - + - Growth at/in: 4°C - - - - - - 37°C + + + + + + 50°C - - - - - NaCl (1%) + + + + + - NaCl (3%) + + + - - - NaCl (4%) - - - - - - pH4 - - - - - - pH6 + + + + + + pH10 + + - + - + Resistance to (µg ml-1): Tetracycline hydrochloride (5)

+ - + - - -

142

Tetracycline hydrochloride (50)

+ - - - - -

Neomycin sulfate (50) + + + + + + Neomycin sulfate (100) - - - - - - Neomycin sulfate (100) - - - - - - Chloramphenicol (5) + - + - - + Chloramphenicol (50) + - - - - + Ampicilin (5) + - + - + - Ampicilin (100) + - - - - - Kanamycin sulfate (5) + - + + - - Kanamycin sulfate (50) + - + - - - Streptomycin sulfate (5) + + + - + + Streptomycin sulfate(50) + - + - - + Erythromycin (5) + + + - + + Erythromycin (100) + - + - + - DNA G+C% mol% (Tm) 60.1 + positive, - negative, w weak Strains: 1 R. pakistanensis BN-19T; 2 R. alkalisoli CCBAU 01393T; 3 R. tarimense PL-41T; 4 R. vignae CCBAU 05176T; 5 R. huautlense SO2T; 6 R. leguminosarum USDA 2370T.

143

BN-19T was compared with sequences of closely related type strains on EzBiocloud

Server database. Strain BN-19T was closely related to R. alkalisoli CCBAU 01393T

showing 97.4% similarity, followed by R. tarimense PL-41T and R. vignae

CCBAU05176T exhibiting 97.3% gene similarity. The sequence similarity of strain

BN-19T with R. huautlese SO2T was 97.2% and 95-96 % to the type strain of the

other species of genus Rhizobium (Table 22).

In the neighbor-joining tree based on 16S rRNA gene sequence (Figure 26),

the strain BN-19T formed a clade comprising species of genus Rhizobium, distinctly

forming a cluster with R. huautlense SO2T, R. alkalisoli CCBAU 01393T, R. vignae

CCBAU 05176T, R. loessense CCBAU 7190BT and R. tarimense. The sequence

similarities of atpD, recA and glnII genes between BN-19T and the reference strains

are given in Table 22. The strain BN-19T was closely related to R. alkalisoli in the

phylogenetic analysis based on atpD, recA and glnII genes with 93.3%, 84% and

89% similarity respectively, supported by the bootstrap value of 97 and 95%

(Fig.27-29).

144

4.7.3 DNA-DNA Hybridization and Chemotaxonomic Analysis

The DNA-DNA relatedness of BN-19T was 15-23% as compared to the reference

strains of the recognized species (Table 22), which is less than the threshold value

of 70% for species delineation (Meier-Kolthoffet al., 2013), confirming that BN-

19T represented a genomic species different from other identified Rhizobium

species. The DNA G+C content of BN-19T was 60.1 % which is within the range

described for the genus Rhizobium (57-66 %) (Young et al., 2001).

145

Rhizobium fabae CCBAU 33202T(DQ835306) Rhizobium phaseoli ATCC 14482T(EF141340)

Rhizobium etli CFN 42T(U28916) Rhizobium leguminosarum LMG 14904T(AM181757)

Rhizobium grahamii CCGE 502T(JF424608) Rhizobium alamiiGBV016T(AM931436)

Rhizobium gallicum R602T(U86343) Rhizobium loessense CCBAU 7190BT(AF364069) Rhizobium hainanenseI66T(U71078)

Rhizobium herbae CCBAU 8301T(GU565534) Rhizobium aggregates IFAM 1003T(X73041)

Rhizobium rubi LMG 156T(X67228) Rhizobium larrymooreiAF3.10T(Z30542) Rhizobium pusenseNRCPB10T(FJ969841)

Rhizobium soli DS 42T(EF363715)Rhizobium pakistanensis BN-19T (AB854065)

Rhizobium tarimense PL-41T(HM371420)Rhizobium cellulosilyticumALA10B2T(DQ855276)

Rhizobium huautlenseS02T(AF025852)Rhizobium alkalisoli CCBAU 01393T (EU074168) Rhizobium vignae CCBAU 05176T(GU128881)

Rhizobium pseudoryzaeJ3-A127T(DQ454123) Bradyrhizobium japonicum USDA 6T (NC017249)

73 59

100

77

9380

98 98

61

60

77 100

52

0.01

Figure 26: Neighbour-joining tree based on nearly complete 16S rRNA gene sequences of strain BN-19T (Rhizobium

pakistanensis sp. nov.) and the recognized species of genus Rhizobium.

139

146

Figure 27:Phylogenetic tree constructed from atpD gene sequences showing the relationship between the novel strain

(Rhizobium pakistanensis sp. nov.) and the recognized Rhizobium species.

R. yanglingense SH 22623T (AY907373)R. gallicum R602spT (AY907371)

R. mongolense USDA 1844T (AY907372)R. sullae IS123T (DQ345069) R. lusitanum P1-7T (DQ431671)

R. mesosinicum CCBAU 25010T (EU120726)R. etli USDA 9032T (AJ294404)

R. multihospitium CCBAU 83401T (EF490019)R. tropici IIB USDA 9030T (AM418789)

R. fabae CCBAU 33202T (EF579929)R. leguminosarum USDA 2370T (AJ294405)

R. soli DS-42T (GQ260191)R. giardinii R4385T (AM418780)

R. tarimense PL-41T (JF508524)R. cellulosilyticum LMG 23643T (AM286429)

R. pakistanensis BN-19T (AB856324)R. huautlense SO2T (AM418782)

R. alkalisoli CCBAU 01393T (EU672461) R. galegae USDA 4128T (AM418779)

92 99

99

53

98

97

63

66

63

79

66

0.01

140

147

Figure 28:Neighbor-joiningphylogenetic tree constructed from recA gene sequences showing the relationship between the

novel strain (Rhizobium pakistanensis sp. nov.) and the recognized Rhizobium species.

R. yanglingense SH 22623T (AY907359) R. gallicum R602spT (AY907357)

R. mongolense USDA 1844T (AY907358) R. alkalisoli CCBAU 01393T (EU672490)R. huautlense SO2T (AM182128) R. giardinii R4385T (AM182123)

R. galegae USDA 4128T (AM182127)R. soli DS-42T (GQ260192)

R. lusitanum P1-7T (DQ431674) R. cellulosilyticum LMG 23642T (AM286427)

R.tropici IIA USDA_9039T (AJ294372) R. multihospitium CCBAU 83401T (EF490029)

R. tropici IIB USDA 9030T (AJ294373)R. mesosinicum CCBAU 25010T (EU120732)

R. etli CFN 42T (CP000133) R. leguminosarum USDA 2370T (AJ294376)

R. fabae CCBAU 33202T (EF579941)R. pisi DSM 30132T (EF113134)R. pakistanensis BN-19T (AB855792)

R. tarimense PL-41T (JF508523)

97

98 100

95

91

69

0.02

141

148

Figure 29:Phylogenetic tree constructed from glnII gene sequences showing the relationship between the novel strain (Rhizobium

pakistanensis sp. nov.) and the recognized Rhizobium species.

R. multihospitium CCBAU 83401T (EF490040)R. tropici IIB USDA 9030T (AF169584)

R. tropici IIA USDA 9039T (AF169583)R. lusitanum P1-7T (EF639841)

R. galegae USDA 4128T (AF169587) R. etli CFN 42T (AF169585)

R. leguminosarum. USDA 2370T (EU155089)R. tibeticum CCBAU 85039T (EU407190)

R. alkalisoli CCBAU 01393T (EU672475)

R. pakistanensis BN-19T (AB856325)R. gallicum USDA 3736T (AY929427)

R. mongolense USDA 1844T (AY929453)S. xinjiangense CCBAU 110T (EU155088)

85

76

69

54

100

0.02

142

149

Table 22: Sequence similarities (%) for 16S rRNA gene, atpD, recA, glnII and DNA-DNA relatedness between

Rhizobiumpakistanensis BN-19T and reference strains.

Strain BN-19T

16S

rRNA atpD recA glnII DNA-DNA relatedness

R. alkalisoli CCBAU 01393T 97.4 93.3 83.5 89.0 20.56

R. tarimense PL-41T 97.3 89.5 80.6 - 20.49

R. vignae CCBAU 05176T 97.3 91.2 85.7 88.8 22.51

R. huautlense SO2T 97.2 93.3 85.4 - 15.91

R. leguminosarum USDA 2370T 96.4 85.8 85.8 86.7 15.58

150

Fatty acid profiling method is commonly used for describing and

characterizing a novel bacterial species (Quan et al., 2005) and for the comparative

assay of profiles that have been acquired under the similar growth conditions

(Logan et al., 2009). The cellular fatty acid compositions of BN-19T and reference

strains are described in Table 23. Thirteen different types of fatty acids were

detected in BN-19T, of which C19:0 cyclo ω8c (22.52%), summed feature 2 (C14:0 3-

OH and/or C16:1 iso 1; 30.37) and summed feature 8 (C18:1 ω7c; 24.07) were the

major fatty acids. Some fatty acids like C16:0, C19:0 cyclo ω8c, summed feature 2, 3

and 8 were found in all tested strains.

Based on all the results obtained, strain BN-19T is considered to represent

anovel species that could be differentiated from closely related Rhizobium species

by

means of biochemical characteristics, DNA-DNA hybridization,

phenotypicproperties and genotypic comparison of 16S rRNA and housekeeping

genes. The name Rhizobium pakistanensis sp. nov. is proposed for this novel

species. The type strain BN-19T (Khalid et al., 2015) is deposited in two

international culture collections i.e., Culture Collection of China Agricultural

University (CCBAU=101086) and Universiteit Gent-Laboratorium voor

Microbiologie (LMG=27895).

151

Table 26: Cellular fatty acid contents (%) of strain BN-19T and type strains of

closely related species of the genus Rhizobium.

Fatty Acid 1 2 3 4 5 6

C12:0 anteiso 0.57 - - 0.14 - C13:0 anteiso 0.48 - - 0.23 0.12 - C14:0 0.64 0.20 - 0.28 0.32 0.28 C16:0 8.84 11.59 9.23 9.92 8.29 4.59 C16:0 iso - - 0.45 - 0.37 C17:1 iso ω5c - - - 0.47 - C17:1 anteiso A 1.90 - - 1.07 - C17:1 anteiso ω9c - 0.98 1.26 - 2.94 C17:0 iso - - - 0.37 - C17:1 ω6c - - - 0.34 0.30 - C17:1ω8c - - - 0.74 - 1.41 C17:0 cyclo 2.91 - - 0.30 0.35 1.26 C17:0 - - - 1.47 0.43 0.37 C17:0 3-OH - - 1.88 1.29 0.26 - C16:0 3-OH 4.05 3.36 0.89 3.99 4.00 2.14 C18:0 0.56 0.55 1.42 0.86 1.27 3.04 C18:0 iso 0.32 0.32 - - 0.23 C18:1 2-OH - 5.06 - 4.48 - C18:0 3-OH - 0.34 1.48 1.71 - 1.63 C18:1ω9c - - - - 0.81 C18:1ω7c 11-methyl - - - 0.27 - 1.72 C18:0 10-methyl - - - - 0.15 C19:0 cyclo ω8c 22.52 4.31 3.61 16.30 2.76 8.93 C20:1ω7c - - - 0.08 - 0.28 Summed Feature 1 - - - 0.41 - 0.77 Summed Feature 2 30.37 14.03 13.51 5.77 14.30 20.0 Summed Feature 3 3.08 2.81 3.19 1.43 1.45 1.45 Summed Feature 8 24.07 55.57 63.08 53.98 59.63 38.74 Strains: 1 R. pakistanensis BN-19T; 2 R. alkalisoli CCBAU 01393T; 3 R. tarimense PL-41T; 4 R. vignae CCBAU 05176T; 5 R. huautlense SO2T; 6 R. leguminosarum USDA 2370T. Summed features are groups of two or three fatty acids that cannot be separated by GLC using the MIDI system. Summed feature 1 comprises C15:1 iso H and/or C13:0

3-OH, summed feature 2 comprises C14:0 3OH/C16:1 iso 1, summed feature 3 comprises C16:1 ω7c and/or C16:1 ω6c, summed feature 8 comprises C18:1ω6c. (-) not detected

152

SUMMARY

The present study was designed with the objectives to isolate bacterial

strains from rhizospheric soil and nodules of groundnut; to characterize and identify

potential bacterial strains by using molecular tagging of 16S rRNA gene

sequencing. Extensive survey was carried out in Pothwar (Attock & Chakwal

districtes) for the collection of rhizospheric soil and nodules. Rhizospheric soil

bacteria were isolated through dilution plate technique using Phosphate Buffer

Saline solution (PBS; 1X) and nutrient media i.e. Tryptic Soya Agar (TSA; Difco).

While, bacteria isolated from nodules were streaked on yeast extract mannitol

(YEM). Total seventy five bacterial strains were isolated and characterized for plant

growth promoting (PGP) traits like P-solubilization, indole acetic acid (IAA) and

nifH amplification. All bacterial strains solubilized substantial quantity of inorganic

phosphate. However, maximum phosphate solubilization (674.1 µg mL-1) was

observed by the inoculation of BN-44 (Pseudomonas oryzihabitans) followed by

BN-55 (Pseudomonas psychrotolerans) which solubilize phosphate up to 610.9 µg

mL-1. The ability to produce IAA in the absence and presence of L-tryptophan (500

µg mL-1) was determined. In the absence of L-tryptophan BN-55 produced

significant amounts of IAA (73.0 µg mL-1). As the concentration of L-tryptophan

was added in the culture, IAA production of was increased up to 97.6 µg mL-1

synthesized by GS-6 (Microbacterium arborescens). Biochemical characterization

was done by using API 20E kit and BIOLOG GN-2 microplate. Resistance of

Rhizobium sp. to seven antibiotics tetracycline hydrochloride, neomycin sulfate,

chloramphenicol, ampicilin, kanamycin sulfate, streptomycin sulfate and

erythromycin at 4 different levels (µg mL-1): 5, 50, 100, 300 was tested.

146

153

Based on plant growth promoting activity, identification of fifty four PGPRs

was performed using robust method of 16S rRNA gene sequence. The strains

belong to seventeen different genera including Bacillus, Pantoea, Pseudomonas,

Arthrobacter, Rhizobium, Microbacterium, Flavobacterium, Acinetobacter,

Kosakonia, Curtobacterium, Variovorax, Stenotrophomonas, Erwinia, Escherichia,

Enterobacter, Chryseobacterium and Kocuria.For bacteria identified as Rhizobium,

PCR amplification and sequencing of housekeeping genes i.e., atpD (510 bp), recA

(530 bp) and glnII (600bp) was also performed.The consensus sequences of each

strain were BLAST using EzBiocloud server database and the phylogenetic position

was determined by analysing their 16S rRNA and housekeeping genes sequence

and comparing it with the known sequences in GenBank database by multiple

sequence alignment with the closest matches performed with the Clustal X

program. Phylogenetic tree construction was performed with Bioedit and Mega 5

software by neighbor joining method with bootstrap values based on 1000

replications.

On the basis of PGP characterization, best characterized PGPRs were

selected for further screening and to evaluate their effectiveness on growth of

groundnut under controlled and field conditions. Most promising strains BN-2

(Bacillus safensis), BN-5 (Arthrobacter globiformis), BN-44 (Pseudomonas

oryzihabitans), BN-55 (Pseudomonas psychrotolerans), GS-2 (Kocuria

turfanensis), GS-4 (Bacillus aryabhattai) and GS-6 (Microbacterium

arborescens) were selected on the basis of plant growth promoting traits to be used

in pot experiment in co-inoculation with Rhizobium sp. The pot experiment was

carried out under greenhouse controlled conditions at PMAS-Arid Agriculture

154

University Rawalpindiduring 2012. The soil used in pot experiments was collected

from the experimental field of PMAS-AAUR. Data regarding growth parameters

and N2-fixation was recorded and result revealed that all selected PGPR strains

significantly increased nodule number, pod yield, biomass yield and N2-fixation of

groundnut over control (uninoculated). On the basis of pot experiment, three

potential PGPRs were selected i.e. BN-55, GS-4 and GS-6 to be evaluated under

field conditions. Field experiment was also carried out at University Research

Farm, Koont during summer 2013. The experiments were designed in randomized

complete block design (RCBD) and replicated three times. Best selected PGPRs

and Rhizobium sp. along with full (N-P @ 20-40 kg ha-1) and half recommended

doze (N-P @ 10-20 kg ha-1) of chemical fertilizers were applied to confirm their

beneficial effect on groundnut yield and N2-fixation. Crop parameters studied

include biomass yield (t ha-1), pod yield (t ha-1), shoot N contents (%) and N2-

fixation. N2-fixation of groundnut was assessed by δ15N natural abundance

technique. The results confirmed an increase in all parameters as compared to

control by combined inoculation of PGPRs and chemical fertilizers.

A Gram-negative, white, rod shaped novel Rhizobium sp. designated as BN-

19T was isolated from groundnut nodules and validated by adopting minimal

standards. The 16S rRNA gene sequence similarities of BN-19T with closely related

type strains R. alkalisoli CCBAU 01393T, R. tarimense PL-41T, R. vignae CCBAU

05176T and R.huautlense SO2T were 97.4, 97.3, 97.3 and 97.2% respectively.

Sequence analysis of housekeeping genes atpD, glnII and recA (with sequence

similarities of ≤92%) and DNA-DNA relatedness between the strain BN-19T and

reference strains (less than 30%) confirmed its position as a novel species in genus

155

Rhizobium. Polyphasic taxonomic experiments were performed to validate the

isolated strain in Rhizobium Research Center, China Agricultural University,

Beijing, China. The DNA G+C content was 60.1 mol%. The dominant fatty acids

of the strain BN-19T were C19:0 cyclo ω8c, summed feature 2 (C14:0 3-OH and/or

C16:1 iso 1) and summed feature 8 (C18:1 ω7c). On the basis of the phylogenetic,

physiological and phenotypic analyses, the strain BN-19T was considered to

represent a novel species of the genus Rhizobium, for which the name Rhizobium

pakistanensis was proposed. The novel strain is deposited in two international

culture collections to be used for scientific community. The

GeneBank/EMBL/DDBJ sequence accession numbers for the partial 16S rRNA,

atpD, glnII and recA gene are AB854065, AB856324, AB856325 and AB855792

respectively.The type strain is BN-19T (=LMG 27895T=CCBAU 101086T).

The findings of this research led us to the conclusion that by considering the

ability of the potential strains BN-55(Pseudomonas psychrotolerans), GS-

4(Bacillus aryabhattai) and GS-6 (Microbacterium arborescens) to solubilze

tricalcium phosphate and synthesize IAA, these strains are confirmed as PGPR

under green house and field experiments. When applied as biofertilzer along with

Rhizobium sp. they significantly increased N2-fixation and yield of groundnut and

proved to be an effective technology. Therefore it could be suggested that farmers

of the rainfed region could raise their profitability by applying Rhizobium and

PGPR biofertilizer along with NP fertilizer @ 20 and 80 kg ha-1. One of the major

challenges to encounter is the use of PGPR for their commercial application. This

encompasses the procedures and practices to maintain the quality, reliability and

productivity of biofertilizer. This large scale production could be achieved either by

156

mixing PGPR with sterilized peat and organic manure or by inoculating the crop

seeds with bacterial innocula. There is a need to design more field studies in

different locations to validate the plant growth promoting effect of the isolated

potential strains in order to use and commercialize them as registered PGPRs.

\

157

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APPENDICES

Appendix 1: ANOVA table for growth of groundnut under greenhouse and field conditions

Greenhouse experiment

SOV df Biomass yield Pod Yield Total shoot N N2-fixed

Treatment 7 F p≥F F p≥F F p≥F F p≥F

Error 16 27.7 0.000 33.4 0.000 31.8 0.000 65.0 0.000

Total 23

Coefficient of variation 2.88% 9.49% 6.83% 4.2%

Filed experiment

SOV df Biomass yield Pod yield Total shoot N N2-fixed

Replication 2 F p≥F F p≥F F p≥F F p≥F

Treatment 5 140.4 0.000 15.33 0.0002 70.9 0.000 71.6 0.000

Error 10

Total 17

Coefficient of variation 3.0% 9.4% 4.39% 10.3%

204

Appendix 2:CCBAU Genbank depoist certificate of BN-19T

205

Appendix 3:LMGGenbank depoist certificate of BN-19T

206

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RABIA KHALID 03-arid-80prr.hec.gov.pk/jspui/bitstream/123456789/6868/1/... · ISOLATION, IDENTIFICATION AND CHARACTERIZATION OF GROUNDNUT RHIZOBACTERIA TO IMPROVE CROP Department