ISOLATION, IDENTIFICATION AND CHARACTERIZATION
OF GROUNDNUT RHIZOBACTERIA TO IMPROVE CROP
YIELD
RABIA KHALID
03-arid-80
Department of Soil Science & Soil and Water Conservation Faculty of Crop and Food Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2015
ii
ISOLATION, IDENTIFICATION AND CHARACTERIZATION
OF GROUNDNUT RHIZOBACTERIA TO IMPROVE CROP
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
Faculty of Crop and Food Sciences
Pir Mehr Ali Shah
Arid Agriculture University Rawalpindi
Pakistan
2015
iii
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: ______
iv
v
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
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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 nif H 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
15 3.9 ASSESSMENT OF N 2-FIXATION BY NATURAL N 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
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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 nif H 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 N 2-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 N 2-Fixation 128
4.7 VALIDATION OF NOVEL SPECIES BN-19 T 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
viii
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 N 2-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 N 2-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-19 T and reference strains 145
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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
18. Phylogenetic tree of BN-52 constructed by neighbor-joining method 97
19. Phylogenetic tree of BN-58 constructed by neighbor-joining method 98
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
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24. Phylogenetic tree of Rhizobium strains for housekeeping gene glnII 103
25. Scanning electron microscopic picture of R. pakistanensis BN-19 T 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
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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 )
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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
T effect on growth and N 2-fixation of groundnut. A novel Rhizobium sp. BN-19 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
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were obtained by PCR amplification using universal primers. For strains identified as Rhizobium housekeeping 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 N 2-fixation. The co- inoculation of bacterial strains along with chemical fertilizer improved yield (76%) and N 2-fixation (193%) under field conditions. Phylogenetic analysis based on 16S rRNA gene sequence revealed that one Rhizobium species BN-19 T, 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-19 T and reference strains (less than
30%), further confirmed that BN-19 T 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 N 2- fixation. Farmers of the rainfed tract could raise their profitability by co-inoculation of PGPR and chemical fertilizer.
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1
Chapter 1 INTRODUCTION
Biological Nitrogen Fixation (BNF) is enzymatic conversion of unavailable molecular nitrogen (~79% N 2) 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
(Nihorimbere et 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 (Qureshi et al ., 2009; Sánchez et al ., 2014). The co-inoculation of effective Rhizobium sp. by PGPR to enhance N 2-fixation by legumes is an approach to increase N 2 supply in eco-friendly agricultural production systems. As a legume,
-1 groundnut is also considered as an efficient N 2-fixer, fixes about 100-130 kg N ha
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 N 2-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
(Chen et al ., 1988) (Table 1). Other N 2-fixing proteobacteria belong to
15
genus Azoarcus are 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 Source of isolation Nodulation Reference isolation 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
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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
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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
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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 + Tan et 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
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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
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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 N 2-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
-4 reservoir. Plants can absorb P in only two soluble forms: monobasic (H 2PO ) and
-4 dibasic ion diabasic (HPO 2 ) (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 Sharma et al ., 2003 reduce catalase activity(33%) in roots. chlorophyll a, chlorophyll b and total chlorophyll contents were significantly enhanced by 34, 48 and 39%, respectively Enterobacter aerogenes Wheat Green house Improve root and shoot length, fresh and Hayat et al ., 2012b and Pseudomonas dry biomass per plant brenneri Thiobacillus sp. and Groundnut Pot and field In pot experiment plant dry weight, Anandham et al ., 2007 Rhizobium sp. nodulation and pod number was increased Under field condition nodule number, plant biomass and pod yield (18%) was augmented Pseudomonas spp. Groundnut Pot and field Increase in pod yield and soil N and P Dey et al ., 2004 contents in pot experiment Under field conditions pod yield was increased (24%) Burkholderia Rice Pot and field Enhanced shoot height (60%), biomass Jha et al ., 2009 cenocepacia , (33%), grain yield (up to 26%) Pseudomonas sp. Serratia sp. and Lettuce Greenhouse Alleviate the soil salinity impact and Han and Lee, 2005 Rhizobium sp improve photosynthesis, mineral contents and plant growth Bacillus species, Sugar beet Green house Increase root weight by 46.7%. Leaf, root Çakmakçi et al ., 2006 Paenibacillus polymyxa , and sugar yield were enhanced by 20.8, Pseudomonas putida and 16.1, and 14.7% respectively Rhodobacter capsulatus Rhizobium tropici , Phaseolus Green house Increase leghemoglobin concentration, Figueiredo et al ., 2007
28
Bacillus sp., vulgaris nitrogenase activity and N 2 fixation Paenibacillus sp. efficiency. Azotobacte r sp. Cotton Field Enhance seed yield (21%), shoot length Anjum et al ., 2007 (5%) and soil microbial population (41 %) Pseudomonas Origanum Green house Shoot length, shoot and root dry weight Banchio et al ., 2008 fluorescens and majorana was enhanced significantly. Oil yield was Bradyrhizobium sp. also increased Bacillus sp. Beta Field Improve yields (5.6-11.0%) Şahin et al ., 2004 vulgaris and Hordeum vulgare Bacillus licheniformis , Barley Green house Increase in root weight by (32.1%) and Çakmakçi et al ., 2007 Rhodobacter capsulatus , shoot weight by (54.2%). Improve uptake Paenibacillus polymyxa , of N, Fe, Mn, and Zn. Soil NO 3-N and P Pseudomonas putida , and contents were increased Bacillus sp. Bacillus firmis , Bacillus Groundnut Pot and field Plant biomass was increased (26%) in pot Kishore et al ., 2005 megaterium and experiment. Improved plant growth and Pseudomonas aeruginosa pod yield (19%) was observed under field condition Bacillus sp ., Lupinas Growth Increased nodulation and plant dry weight Lucas Garcia et al ., Pseudomonas sp. and albus chamber 2004 Bradyrhizobium japonicum Pseudomonas putida, Mung bean Axenic and in Increased nodulation and suppression of Shaharoona et al ., Pseudomonas fluorescens vitro ethylene 2006 and Bradyrhizobium sp . Azospirillum brasilense Soybean and Growth Increased seed germination and nodule Cassàn et al ., 2009 and Bradyrhizobium corn chamber formation japonicum Bacillus subtilis and Soybean Green house Augmentation of mass and nodule number, Bai et al ., 2003 Bradyrhizobium and field biomass, nitrogen contents and grain yield
29
japonicum Bacillus subtilis and Tomato In vitro Regulate and increase root growth and Felici et al ., 2008 Azospirillum brasilense development Pseudomonas aurantiaca Wheat and Field Improve microbial colonization in root and Rosas et al ., 200 8 maize increase yield Pseudomonas sp . and Wheat In vitro Increase root elongation (17.3%), root dry Khalid et al ., 2004 Bacillus sp. weight (13 .5%), shoot elongation (37 .7%) and shoot dry weight (36.3%) Bacillus sp. Raspberry Field Enhanced yield (33.9%), cluster number Orhan et al ., 2006 (28.7%), berries number (36.0%). Increase soil P contents Mung bean, Axenic Biomass and grain yield in wheat (45%) Hayat et al ., 2013 mash bean and beans (50%) are improved and wheat significantly Kluyvera ascorbata Tomato, Axenic Minimize the deleterious effects of nickel, Burd et al ., 2000 canola and zinc and lead by synthesizing siderophores mustard Bacillus spp. Tomato, bell Green house Protection from leaf -spotting fungal and Kloepper et al ., 2004 a pepper, bacterial pathogens, systemic viruses, watermelon, crown-rotting fungal pathogen, root-knot sugar beet, nematodes, and a stem-blight fungal tobacco, Arab pathogen in addition to damping-off, blue idopsis sp. mold, and late blight diseases Pinus Axenic Stimulated growth and associative nitrogen Chanway and Holl, contorta fixation 1991 Pseudomonas putida Canola Axenic Enhanced root elongation and the plants Sun et al ., 1995 were able to grow at 5 °C Pseudomonas sp. Wheat In vitro Plant height, root and shoot biomass and de Freitas and number of tillers was increased Germida, 1990 Mesorhizobium sp. Chickpea P and Iron (Fe) uptake was enhanced. Verma et al ., 2014 and Pseudomonas Minimize the deleterious effects of wilt aeruginosa and root rot. Increased nodulation (86%),
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
-3 ions i.e., PO 4 , 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 (Khan et 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 was Rhizobium (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 N 2-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 NH 3 (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 (C 20 ) or monocarboxylic (C 19 ). 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 GA 1, GA 4, GA 9 and GA 20 is revealed in Rhizobium meliloti by Atzorn et al . (1988), GA 1, GA 3, GA 9, GA 19 and
GA 20 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 N 2-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 N 2 to NH 3 . 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
(Gaiero et 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 ( Brear et 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 N 2-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 ( Ohyama et 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. (Rokhzadi et 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.
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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 N 2-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
Isolation
Incubation at 28°C
Series of re-isolation steps
Incubation at 28°C
Pure bacterial isolates
Figure 3: Schematic diagram for the isolation of bacteria from groundnut
rhizospheric soil.
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
KH 2PO 4 0.25g
K2HPO 4 0.25g
NaCl 0.1g
MgSO 4.7H 2O 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 (C 2HCl 3O2 0.5M) 40.9 g), 400µL reagent B (0.01M ammonium molybdate 6.2g) and 1000 µL reagent C (sodium citrate (Na 3C6H5O7.2H 2O 0.1 M)
29.4g + sodium arsenite (NaAsO 2 0.2M) 26g + (5%) glacial acetic acid (99.9%) 50 mL) was added. Standards were prepared by KH 2PO 4 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 FeCl 3 solution). The appearance of pinkish color in thesolution indicated IAA production ability of the isolates.
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Table 4: Composition of Nautiyal media.
Nautiyal media Quantity g L -1 pH 7 Glucose 10
(NH 4)2SO 4 0.5
MgSO 4.7H 2O 0.1 KCl 0.2 NaCl 0.2
FeSO 4.7H 2O 0.002
MnSO 4. H 2O 0.002
Ca 3(PO 4)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 nifH Gene
The ability of the bacterial isolates for nitrogen fixing nifH gene was determined through PCR (Figure 4) using reverse and forward nif H 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 Na2CO 3 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 5ʹTGCGAYCCSAARGCBGACTC3 ʹ PolR b 5ʹATSGCCATCATYTCRCCGGA3 ʹ 16S rRNA 1510R 5ʹ-GGCTACCTTGTTACGA-3ʹ 9F 5ʹ-GAGTTTGATCCTGGCTCAG-3ʹ (P6) 5ʹ-TACGGCTACCTTGTTACGACTTCACCCC -3ʹ (P1) 5ʹ-AGAGTTTGATCCTGGCTCAGAACGAACGCT - 3ʹ atpD atpD 255F 5ʹ-AACGTC GCTSGGCCGCATCMTS -3ʹ atpD 782R 5ʹ-GCCGACACTTCMGAACCNGCCTG -3ʹ recA recA-F6 5ʹ-CGKCTSGTAGAGGAYAAATCGGTGGA-3ʹ recA -555 5ʹ-CGRATCTGGTTGATGAAGATCACCAT -3ʹ glnII glnII 12F 5ʹ-YAAGCTCGAGTACAT YTGGCT-3ʹ glnII 689R 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
Initial Denaturation 95 oC, 3min
Denaturation 94 oC, 1min
Annealing 58 oC, 1min x 30 cycles
Elongation 72 oC, 1min
Final Exrension 72 oC, 6min
Figure 5: PCR cycling conditions for 16S rRNA and atpD gene amplification.
59
Initial Denaturation 95 oC, 5min
Denaturation 94 oC, 1min
Annealing 55 oC, 1min x 30 cycles
Elongation 72 oC, 1min
Final Exrension 72 oC, 10min
Initial Denaturation 95 oC, 5min
Denaturation 94 oC, 1min
Annealing 58 oC, 1min x 30 cycles
Elongation 72 oC, 1min
Final Exrension 72 oC, 10min
Figure6: PCR cycling conditions for recA and glnII gene amplification.
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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 N 2-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% H 2O2, 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 10 9 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
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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 N 2-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 N 2-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 m 2. 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 m 2 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 65 oC for 48 hrs and grinded by usingWiley Mill. Samples were stored in plastic containers for the determination of
15 N contents. For N 2-fixation by natural N 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 for 15 N 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 H 2O2). 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).
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15 3.9 ASSESSMENT OF N 2-FIXATION BY NATURAL N ABUNDANCE
The %Pfix (proportion of N derived from air) was estimated using the following equation.
%Pfix = 100 x 15 N (soil N) - 15 N (legume N)
( 15 N (soil N) –B)
Where:
15 N (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).
15 B is the N of the same N 2 fixing plants when grown with N 2 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-19 T), exhibited < 97% similarity with its closely related identified strains was considered as potential novel candidate and validated by
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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-
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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
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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 10 5 D. Fragmented DNA was diluted with the help of 0.1X SSC liquor to obtain concentration around A260= 1.5 ~ 2.0.
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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 VA *VB 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
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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
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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 nif H 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 N 2-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
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plant growth promoting attributes including indole-3-acetic acid (IAA) production, phosphate solubilization, nif H 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
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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 nifH gene 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 (Raymond et al ., 2004). Symbiotic bacteria
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includinggenus Rhizobium and many free living soil bacteria including
Azospirillum ,
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Table 8: Plant growth promoting traits of strains isolated from rhizospheric soil and nodules of groundnut.
S. No. Strain ID Location IAA with IAA P Catalase Oxidase nif H Gram tryptophan without solubilization amplification staining trytophan ------(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 + + - +
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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 61 1 ± 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 + + - +
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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-Kishi et 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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Azotobacter , Klebsiella ,Rhodospirillum ,Azotobacter , Clostridium , Azospirillum ,
Burkholderia , Azoarcusand Rhodobacter have been reported to possess nifH gene and are involved in the process of N 2-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 N 2-fixing bacteria identification at species or genus level.
Moreover, nifH gene sequence balance could provide new insights into the genetic diversity of N 2-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
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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 , rpoB and thrC can 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. Closely related Taxa identified by BLAST search using ezbiocloud.net Source of Sr. # Strain ID Location Highest similarity of isolation Species DDBJ Accession 16SrRNA gene sequence % 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
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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
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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 atpD recA glnII DDBJ Accession Number identification 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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50 GS -5(LC040955) GS -1 (AB773829) 52 Bacillus subtilis subsp. spizizenii NRRL B-23049 T (CP002905 ) 48 Bacillus tequilensis KCTC 13622 T (AYTO01000043 ) BN -10 (LC038164), BN -14 (LC038165) , BN -49 (LC040945) Bacillus subtilis subsp. subtilis NCIB 3610 T (ABQL01000001 ) 56 74 BN -26 (LC040931) Bacillus amyloliquefacienssubsp. plantarum FZB42 T (CP000560) 99 Bacillus siamensis KCTC 13613 T (AJVF01000043) 55 Bacillus amyloliquefaciens subsp. amyloliquefaciens DSM 7 T (FN597644) Bacillus atrophaeus JCM 9070 T (AB021181)
76 BN -2 (AB971370), BN -17 (LC038167), BN -28 (LC040933) 62 Bacillus safensis FO -36b T (ASJD01000027) 88 Bacillus pumilus ATCC 7061 T (ABRX01000007) Bacillus invictus Bi.FFUP1 T (JX183147) T 100 Bacillus stratosphericus 41KF2a (AJ831841) Bacillus altitudinis 41KF2b T (ASJC01000029 ) 99 Bacillus aerophilus 28K T (AJ831844 ) Bacillus xiamenensis HYC-10 T (AMSH01000114) Brevibacillus brevis (D78457)
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
Pantoea vagans LMG 24199 T (EF688012) Pantoea brenneri LMG 5343 T (EU216735) Pantoea conspicua LMG 24534 T (EU216737 ) BN -3 (AB971371) 63 Curtobacterium plantarum CIP 108988 T (JN175348) T Pantoea agglomerans DSM 3493 (AJ233423 ) Pantoea eucalypti LMG 24198 T (EF688009 )
Pantoea anthophila LMG 2558 T (EF688010 ) 73 T 99 Pantoea deleyi LMG 24200 (EF688011) Pantoea ananatis LMG 2665 T (JMJJ01000010 ) T 74 Flavobacterium acidificum LMG 8364 (JX986959 ) 55 Pantoea allii LMG 24248 T (AY530795) Pantoea stewartii subsp. stewartii LMG 2715 T (Z96080 ) 99 Pantoea stewartii subsp. indologenes LMG 2632 T (Y13251) Pantoea septica LMG 5345 T (EU216734 ) Pectobacterium carotovorum (AJ233411)
0.005
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.
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100 BN -31 (LC040936) BN -32 (LC040937), BN -38 (LC040939) 99 Pseudomonas azotoformans IAM1603 T (D84009) T Pseudomonas fragi ATCC 4973 (AF094733) Pseudomonas chengduensis MBRT/EU307111 T 100 Pseudomonas toyotomiensis HT-3 (AB453701) T 66 Pseudomonas oleovorans subsp. lubricantis RS1 (DQ842018) 40 Pseudomonas tuomuerensis 78-123 T (DQ868767) Pseudomonas stutzeri ATCC 17588 T (CP002881) Pseudomonas benzenivorans DSM 8628 T (FM208263)
T 36 Pseudomonas flavescens B62 (U01916) Pseudomonas luteola IAM 13000 T (D84002) BN -46 (LC040944 ) BN -43 (LC040941) BN -44 (LC040942) Pseudomonas oryzihabitans IAM 1568 T (AM262973) 100 BN -55 (LC040947 ) BN -47 (AB773830) BN -4 (AB971372) Pseudomonas psychrotolerans C36 T (AJ575816) Acenitobacter lwoffii (U10875)
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 .
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92
T 77 Arthrobacter polychromogenes DSM 20136 (X80741) 99 Arthrobacter oxydans DSM 20119 T (X83408) 62 Arthrobacter scleromae YH-2001 T (AF330692) 82 Arthrobacter siccitolerans 4J27 T (GU815139) Arthrobacter phenanthrenivorans Sphe3 T (CP002379) 65 T 87 Arthrobacter niigatensis LC4 (AB248526) Arthrobacter defluvii 4C1-aT (AM409361) 88 T Arthrobacter chlorophenolicus A6 (CP001341) T 54 Arthrobacter equi IMMIB L-1606 (FN673551) 60 Arthrobacter enclensis NIO-1008 T (JF421614) BN -5 (AB971373) T 100 Arthrobacter oryzae KV-651 (AB279889) 71 Arthrobacter humicola KV-653 T (AB279890) T 78 Arthrobacter globiformis NBRC 12137 (BAEG01000072) T 99 Arthrobacter pascens DSM 20545 (X80740) Arthrobacter flavus JCM 11496 T(AB299278) Arthrobacter crystallopoietes DSM 20117 T (X80738) Psudomonas putidia (AJ007910)
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.
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93
BN -12 (AB969783) T 100 Rhizobium pusense NRCPB10 (FJ969841)
93 BN -23 (AB969785 ) BN -39 (AB969787 ) 59 Rhizobium vitis LMG 8750 T(X67225) BN -25 (AB969786 ) 43 100 Rhizobium herbae CCBAU 83011 T(GU565534) Rhizobium leguminosarum LMG 14904 T(AM181757) T 100 Rhizobium etli CFN 42 (U28916) 43 59 Rhizobium phaseoli ATCC 14482 T(EF141340) T 57 Rhizobium pakistanensis BN -19 (AB854065) Rhizobium tarimense PL -41 T(HM371420) T 19 Rhizobium soli DS-42 (EF363715) Rhizobium cellulosilyticum ALA10B2 T(DQ855276) 32 T 70 Rhizobium alkalisoli CCBAU 01393 (EU074168) 92 Rhizobium vignae CCBAU 05176 T(GU128881) 99 BN -20 (AB969784 ) T 62 Rhizobium huautlense S02 (AF025852) 42 BN -50 (AB969788 ) 71 BN -6(AB969782) Rhizobium oryzae Alt 505 T (EU056823) 99 Rhizobium pseudoryzae J3-A127 T(DQ454123) Bradyrhizobium japonicum USDA 6 T (NC 017249)
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 6 T was used as outgroup.
90 94
100 BN -8 (LC038163) T 51 Microbacterium barkeri DSM 20145 (X77446) Microbacterium indicum BBH6 T (AM158907) 48 52 Microbacterium oryzae MB10 T (GU564360) Microbacterium luticocti DSM 19459 T (AULS01000007) 95 45 Microbacterium immunditiarum SK 18 T (DQ119293) T 99 Microbacterium marinilacus YM11-607 (AB286020) Microbacterium paludicola US15 T (AJ853909) 97 Microbacterium radiodurans GIMN1.002 T (GQ329713) 95 Microbacterium arborescens DSM 20754 T (X77443) T 88 Microbacterium natoriense TNJL143-2 (AY566291) BN -24 (LC040930) T 63 Microbacterium azadirachtae AI -S262 (EU912487) 69 Microbacterium kyungheense THG-C26 T (JX997973) T Microbacterium marinum H101 (HQ622524) T 83 Microbacterium arthrosphaerae CC -VM -Y (FN870023) 92 Microbacterium jejuense THG-C31 T (JX997974) Leucobacter aerolatus (FN597581)
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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T 75 Curtobacterium albidum DSM 20512 (AM042692 ) 45 Curtobacterium citreum DSM 20528 T (X77436) 32 Flavobacterium oceanosedimentum ATCC 31317 T (EF592577)
30 BN -16 (LC038166 ) 40 Curtobacterium pusillum DSM 20527 T (AJ784400) 100 Curtobacterium luteum DSM 20542 T (X77437) Curtobacterium ammoniigenes NBRC 101786 T (AB266597) T 36 Curtobacterium herbarum P 420/07 (AJ310413 ) 47 Curtobacterium flaccumfaciens LMG 3645 T (AJ312209 ) T 100 Mycetocola tolaasinivorans CM-05 (AB012646) Mycetocola lacteus CM-10 T (AB012648 ) 83 Compostimonas suwonensis SMC46 T (JN000316 ) 51 Frondihabitans sucicola GRS42 T (JX876867 ) Brevibacterium linens DSM 20425 T (X77451)
0.01
Figure 13: Dendogram of BN-16 derived from 16S rRNA gene sequence constructed by neighbor-joining method.
Brevibacterium linens DSM 20425 T was used as outgroup.
92 96
T 99 Acinetobacter johnsonii CIP 64.6 (APON01000005) 53 Acinetobacter oryzae B23 T (GU954428) Acinetobacter bouvetii DSM 14964T/APQD01000004 Acinetobacter kyonggiensis KSL5401-037 T (FJ527818) Acinetobacter beijerinckii CIP 110307 T (APQL01000005) T Acinetobacter kookii 11-0202 (JX137279) Acinetobacter gyllenbergii CIP 110306 T (ATGG01000001) Acinetobacter parvus DSM 16617 T (AIEB01000124)
T 64 Acinetobacter tjernbergiae DSM 14971 (ARFU01000016) Acinetobacter venetianus RAG-1T (AKIQ01000085) Prolinoborus fasciculus CIP 103579 T (JN175353) GS -7 (AB777645) 100 Acinetobacter pittii (HQ180184) Acinetobacter indicus CIP 110367 T (KI530754)
96 BN -22 (AB777646) Acinetobacter junii CIP 64.5 T (APPX01000010) 62 Acinetobacter baumannii ATCC 19606 T (ACQB01000091) 67 Acinetobacter gerneri DSM 14967 T (APPN01000041) Psychrobacter immobilis (U39399)
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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97
T 99 Escherichia coli O157 EC4115 (CP001164) 43 Escherichia fergusonii ATCC 35469 T (CU928158) T Cedecea lapagei GTC 346 (AB273742) Citrobacter farmeri CDC 2991-81 T (AF025371) Salmonella enterica subsp. enterica LT2 T (AE006468) 41 97 Salmonella enterica subsp. salamae DSM 9220 T(EU014685) Enterobacter cloacae subsp. cloacae ATCC 13047 T (CP001918) Escherichia hermannii GTC 347 T (AB273738) 50 Kosakonia cowanii CIP 107300 T (AJ508303) 84 90 BN -30 (LC040935) 97 BN -29 (LC040934) Kosakonia oryzae Ola 51 T (EF488759) Enterobacter hormaechei ATCC 49162 T (AFHR01000079) 87 Enterobacter ludwigii DSM 16688 T (AJ853891) Sodalis glossinidius M1 T (M99060)
0.005
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 M1 T was used as outgroup.
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BN -33 (LC040938) Pantoea conspicua LMG 24534 T (EU216737 ) Curtobacterium plantarum CIP 108988 T (JN175348 ) T 43 Pantoea anthophila LMG 2558 (EF688010) Pantoea vagans LMG 24199 T (EF688012) Pantoea agglomerans DSM 3493 T (AJ233423 ) 23 Pantoea brenneri LMG 5343 T (EU216735) Pantoea deleyi LMG 24200 T (EF688011) Pantoea ananatis LMG 2665 T (JMJJ01000010) 43 T 24 Pantoea stewartii subsp. indologenes LMG 2632 (Y13251 ) T 91 Pantoe a stewartii subsp. stewartii LMG 2715 (Z96080) 40 Pantoea allii LMG 24248 T (AY530795 ) T 41 28 Flavobacterium acidificum LMG 8364 (JX986959 ) T 29 Pantoea eucalypti LMG 24198 (EF688009) Pantoea gaviniae A18/07 T (GQ367483) Pantoea septica LMG 5345 T (EU216734 ) Pantoea rwandensis LMG 26275 T (JF295055) Brevibacterium linens DSM 20425 T (X77451 )
0.02
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 20425 T was used as outgroup.
95
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T 74 Erwinia amylovora NBRC 12687 (BAYW01000035) 85 Erwinia pyrifoliae DSM 12163 T (FN392235) 93 Erwinia uzenensis YPPS 951 T (AB546198 ) 81 Erwinia piriflorinigrans CFBP 5888 T (GQ405202 ) T 52 Erwinia tasmaniensis Et1/99 (CU468135 ) Erwinia rhapontici ATCC 292 83T/U80206
33 43 Candidatus Erwinia dacicolaBactrocera oleae (AJ586620) 96 BN -45 (LC040943) 39 99 Erwinia persicina ATCC 35998 T (U80205 ) Erwinia billingiae Eb661 T (AM055711 ) 47 Erwinia toletana CECT 5263 T (FR870447 ) T 30 Pantoea eucrina LMG 2781 (EU216736) 98 Pantoea wallisii LMG 26277 T (JF295057) Pantoea rodasii LMG 26273 T (JF295053) 58 Pantoea rwandensis LMG 26275 T (JF295055 ) Pantoea septica LMG 5345 T (EU216734) Brenneria alni ICMP 12481 T (AJ223468)
0.005
Figure 17: Neighbour-joining phylogenetic tree confirming the position of strain BN-45 in genus Erwinia . Brenneria alni ICMP 12481 T was used as outgroup.
96 100
T 97 Enterobacter cancerogenus LMG 2693 (Z96078) T 25 Enterobacter asburiae JCM 6051 (AB004744 ) Enterobacter ludwigii DSM 16688 T (AJ853891 ) 12 88 Leclercia adecarboxylata GTC 1267 T (AB273740 ) Enterobacter xiangfangensis 10-17 T (HF679035 ) 40 Enterobacter hormaechei ATCC 49162 T (AFHR01000079) BN -52 (LC040946 ) 100 Escherichia vulneris ATCC 33821 T (AF530476 ) T 99 Klebsiella michiganensis W14 (JQ070300 ) Klebsiella oxytoca JCM 1665 T (AB004754) T 63 Enterobacter aerogenes KCTC 2190 (CP002824) T 56 Yokenella regensburgei GTC 1377 (AB273739) T 30 Citrobacter freundii ATCC 8090 (ANAV01000046 ) 97 Citrobacter murliniae CDC 2970-59 T (AF025369)
0.001
Figure 18: Dendogram of BN-52 derived from 16S rRNA gene sequence constructed by neighbor-joining method.
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Leclercia adecarboxylata GTC 1267 T (AB273740) T 28 Enterobacter ludwigii DSM 16688 (AJ853891) Enterobacter kobei CIP 105566 T (AJ508301) 29 Enterobacter cloacae subsp. dissolvens LMG 2683 T (Z96079) 33 BN -58 (LC040950) Enterobacter hormaechei ATCC 49162 T (AFHR01000079) Erwinia aphidicola DSM 19347 T (AB273744) 30 9 Citrobacter freundii ATCC 8090 T (ANAV01000046) 61 T 79 Citrobacter murliniae CDC 2970-59 (AF025369) T 34 70 Citrobacter youngae CECT 5335 (AJ564736) Salmonella enterica subsp. enterica LT2 T (AE006468) Enterobacter cloacae subsp. cloacae ATCC 13047 T (CP001918) 66 T 30 Klebsiella oxytoca JCM 1665 (AB004754) T 95 Klebsiella michiganensis W14 (JQ070300) Cedecea lapagei GTC 346 T (AB273742) Enterobacter oryzendophyticus REICA 082 T (JF795011) Buchnera aphidicola (M63246)
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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T 98 Chryseobacterium vietnamense GIMN1.005 (HM212415) 52 Chryseobacterium bernardetii NCTC 13530 T (JX100816) 63 Chryseobacterium nakagawai NCTC 13529 T (JX100822) T 6 Chryseobacterium aquifrigidense CW9 (EF644913) GS -3 (LC040953) T 308 Chryseobacterium gleum ATCC 35910 (ACKQ01000057) T Chryseobacterium arthrosphaerae CC-VM-7 (FN398101) T Chryseobacterium indologenes NBRC 14944 (BAVL01000024) 60 Chryseobacterium tructae 1084-08 T (FR871429) T 94 Chryseobacterium contaminans C26 (KF652079) 61 Chryseobacterium artocarpi UTM-3T (KF751867) Chryseobacterium oncorhynchi 701B-08 T (FN674441) T 43 Chryseobacterium viscerum 687B-08 (FR871426) 86 Chryseobacterium lactis NCTC 11390 T (JX100821) Chryseobacterium oranimense H8 T (EF204451) Flavobacterium aquatile (JX657053)
0.02
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.
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T 66 Kocuria himachalensis K07-05 (AY987383) 62 Kocuria rosea DSM 20447 T (X87756) 69 Kocuria polaris CMS 76 T (AJ278868) T 48 Kocuria aegyptia YIM 70003 (DQ059617) Kocuria sediminis FCS-11 T (JF896464) 70 95 GS -2(LC040952) T 81 Kocuria turfanensis HO-9042 (DQ531634) 60 Kocuria flava HO-9041 T (EF602041) Kocuria marina KMM 3905 T (AY211385) T 97 Kocuria gwangalliensis SJ2 (EU286964) T 98 Kocuria atrinae P30 (FJ607311) Kocuria assamensis S9-65 T (HQ018931) T 100 Kocuria palustris DSM 11925 (Y16263) Micrococcus luteus (AJ536198)
0.005
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.
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BN-20 (AB970799) BN-50 (LC061205) 100 BN-25 (LC061203) BN-12 ( LC061201) 53 R. huautlense (AM418782 ) BN-39 ( LC061204) BN-6 ( LC061200) 52 80 R. pakistanensis BN-19 T (AB856324) 100 BN-23 ( LC061202) 45 R. alkalisoli LMG24763 T(EU672461) 71 R. galegae (AM418779 ) R. cellulosilyticum (AM286429 )
R. soli DS-42 T (GQ260191) R. yanglingense SH22623 T(AY907373) R. multihospitium CCBAU 83401 T(EF490019) 96 R. tropici LMG 9503 T(AM418789) R. etli CFN 42 T (CP000133) T 56 R. fabae CCBAU 33202 (EF579929) 96 R. leguminosarum (AJ294405)
0.01
Figure 22: Phylogenetic tree of isolated Rhizobium strains based on housekeeping gene sequence of atpD gene.
101 105
BN-50 (LC061210) 100 BN-6 (AB971367) 97 BN-25 (LC061208) 99 R. alkalisoli LMG24763 T(EU672490) R. huautlense (AM182128 ) R. giardinii (AM182123 ) R. galegae (AM182127 ) R. soli DS-42 T (GQ260192) R. etli CFN 42 T T 69 R. fabae CCBAU 33202 (EF579941) 86 R. leguminosarum USDA 2370 T (AJ294376) T 50 R. cellulosilyticum LMG 23642 (AM286427) R. tropici A USDA 9039 T(AJ294372) R. tropici B USDA 9030 T(AJ294373) T 100 R. pakistanensis BN-19 (AB855792) BN-20 (AB970801 BN-23 (LC061207) 100 BN-12 ( LC061206 ) 98 BN-39 (LC061209)
0.02
Figure 23: Phylogenetic tree of isolated Rhizobium strains based on housekeeping gene sequence of recA gene constructed by neighbor-joining method.
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96 BN-25 (LC061213) 85 BN-6 (AB971366) 100 BN-50 (LC061215) 56 R. alkalisoli LMG24763 T(EU672475) 27 BN-20 (AB970800) R. gallicum (AY929427)
24 47 BN-23 (LC061212) 100 BN-39 (LC061214) 70 BN-12 (LC061211) R. pakistanensis BN -19 T (AB856325) 70 R. etli (AF169585) 33 R. leguminosarum (EU155089) R. galegae (AF169587) 18 R. lusitanum (EF639841)
71 R. tropici (AF169583) T 54 R multihospitium CCBAU 83401 (EF490040) 73 R. tropici (AF169584) S. xinjiangense (EU155088 )
0.02
Figure 24: Phylogenetic tree of isolated Rhizobium strains based on housekeeping gene sequence of glnII gene constructed by neighbor-joining method.
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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
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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.
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) - - + - - - - - + Positive; - negative
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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 H 2S 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.
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) - - - - - + + Mannitol (MAN) - - - - + + + Inositol (INO) ------Sorbitol (SOR) - - - + + + - Rhamnose (RHA) - - - + + - - Saccharose (SAC) - - - - + + + Melibiose (MEL) - - - - + + +
Amygdalin (AMY) assimilation Sugars - - - - + + + Arabinose (ARA) - - + + + - + + Positive, - negative
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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 Accession P- IAA Oxidase Catalase nifH Highest Similarity (%) ID number solubilization Production gene 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-Kishi et 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 N 2-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 Pod Biomass No Yield 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 N 2 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 N 2- 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 N 2-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. aryabhattai proved to be the promising PGPR for enhancing the symbiotic activity of Rhizobium sp. The percent increase in N 2-fixed by T5, T7 and
T8 is 150% as compared to T1 (control) which fixed only 6 mg g -1 of nitrogen.
-1 Minimum N 2 (8 mg g ) 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 N 2-fixation by Rhizobium application owing to their potential of fixing atmospheric nitrogen.
Treatments Shoo t N Ndfa Shoot N N2-fixed uptake ------%------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
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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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 N 2 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. and Azospirillum spp. on rhizobium-legume symbiotic effectiveness (Bashan et al .,2004; Valverde et al ., 2006). The combined inoculation of groundnut seed with Rhizobium 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
%N dfa to 42 and 49% as compared to control. This difference in N 2-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 N 2-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
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Table 18: Percent increase in yield and N 2-fixation of groundnut over control
under pot experiment.
Treatments Nodule Pod Yield Biomass Ndfa Shoot N N2-fixed No Yield uptake 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 Biomass Pod yield density 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
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.
PGPRs: (BN-55: Pseudomonas psychrotolerans + GS4: Bacillus aryabhattai + GS-6: Microbacterium arborescens )
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(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 .and Pseudomonas 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).
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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
-1 N application at the rate of 40 kg ha decreased nodulation and N 2-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 N 2-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,
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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 N 2-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 N 2-Fixation of Groundnut
Efficacy of nitrogen fixation ability is indicated by accumulation of N 2 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
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Table 20: Effect of co- inoculation of effective PGPR and Rhizobium sp . with and without NP fertilizer on N 2-fixation of groundnut under field experiment.The significant increase in N 2-fixation as compared to control is attributed to the potential of Rhizobium sp . to fix atmospheric nitrogen.
Treatments Shoot N Ndfa Shoot N N2-fixed content uptake ------%------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
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.
PGPRs: (BN-55: Pseudomonas psychrotolerans + GS4: Bacillus aryabhattai + GS-6: Microbacterium arborescens )
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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 N 2- 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
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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 (%N dfa ) are similar to the results of N-fixed (Table 20), exhibiting a significant effect of PGPR and
Rhizobium inoculation along with chemical fertilizers on %N dfa . 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 %N dfa from N fixation ranged between 45 and 54% by using the 15 N natural abundance technique.
They further concluded that under conditions of relatively high plant available 15 N conditions in the soil, the natural abundance technique is a feasible method to access the N 2-fixation.
4.7 VALIDATION OF NOVEL SPECIES FROM GROUNDNUT
NODULES ASRhizobium pakistanensis BN-19 T
Among the eight Rhizobium sp . isolated from groundnut nodules, one strain
(BN-19 T) formed a distinct 16S rRNA gene type by showing less than 97% 16S
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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-19 T, polyphasic taxonomic experiments were performed in Rhizobium Research Center, China Agricultural University, Beijing,
China. Five reference strains of the genus Rhizobium were used namely R. alkalisoli
CCBAU 01393 T, R. tarimense PL-41 T, R. vignae CCBAU 05176 T, R. huautlense
SO2 T, and R. leguminosarum USDA 2370 T for DNA-DNA relatedness, fatty acid analysis, physiological and biochemical tests. These reference strains were compared with BN-19 T under the same laboratory conditions. Considering the sequence analysis of 16S rRNA and housekeeping genes, the strain BN-19 T 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-19 T (=LMG 27895 T=CCBAU 101086 T) 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-19 T 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
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shaped, 0.4-1.03 µm long and 0.3-1.2 µm wide (Figure 25). The phenotypic characterization of the strain BN-19 T in comparison to the reference strains are presented in Table 21. The results depicted that 69 features differentiate BN-19 T from its closely related strains, whereas 26 were common to all tested strains. BN-
19 T 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-19 T 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-19 T and reference strains is depicted in Table 21.
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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-19 T exhibiting the rod shaped structure of Rhizobium .
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Table 21:Differential features for type strains of R. pakistanensis BN-19 T 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- + - - + w w methyl-ester 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 -
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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)
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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(5 0) + - + - - + Erythromycin (5) + + + - + + Erythromycin (100) + - + - + - DNA G+C% mol% ( Tm) 60.1
+ positive, - negative, w weak
Strains: 1 R. pakistanensis BN-19 T; 2 R. alkalisoli CCBAU 01393 T; 3 R. tarimense PL-41 T; 4 R. vignae CCBAU 05176 T; 5 R. huautlense SO2 T; 6 R. leguminosarum USDA 2370 T.
143
BN-19 T was compared with sequences of closely related type strains on EzBiocloud
Server database. Strain BN-19 T was closely related to R. alkalisoli CCBAU 01393 T showing 97.4% similarity, followed by R. tarimense PL-41 T and R. vignae
CCBAU05176 T exhibiting 97.3% gene similarity. The sequence similarity of strain
BN-19 T with R. huautlese SO2 T 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-19 T formed a clade comprising species of genus Rhizobium , distinctly forming a cluster with R. huautlense SO2 T, R. alkalisoli CCBAU 01393 T, R. vignae
CCBAU 05176 T, R. loessense CCBAU 7190B T and R. tarimense . The sequence similarities of atpD , recA and glnII genes between BN-19 T and the reference strains are given in Table 22. The strain BN-19 T 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-19 T 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-Kolthoff et al ., 2013), confirming that BN-
19 T represented a genomic species different from other identified Rhizobium species. The DNA G+C content of BN-19 T was 60.1 % which is within the range described for the genus Rhizobium (57-66 %) (Young et al ., 2001).
145
T 73 Rhizobium fabae CCBAU 33202 (DQ835306 ) 59 Rhizobium phaseoli ATCC 14482 T(EF141340) Rhizobium etli CFN 42 T(U28916) Rhizobium leguminosarum LMG 14904 T(AM181757) T Rhizobium grahamii CCGE 502 (JF424608) Rhizobium alamii GBV016 T(AM931436) 77 T Rhizobium gallicum R602 (U86343) 100 Rhizobium loessense CCBAU 7190B T(AF364069) 61 Rhizobium hainanense I66 T(U71078) Rhizobium herbae CCBAU 8301 T(GU565534) Rhizobium aggregates IFAM 1003 T(X73041) 98 Rhizobium rubi LMG 156 T(X67228) 98 T 80 Rhizobium larrymoorei AF3.10 (Z30542) 93 Rhizobium pusense NRCPB10 T(FJ969841) Rhizobium soli DS 42 T(EF363715) T 60 Rhizobium pakistanensis BN-19 (AB854065) Rhizobium tarimense PL -41 T(HM371420) T 52 Rhizobium cellulosilyticum ALA10B2 (DQ855276) Rhizobium huautlense S02 T(AF025852) 100 T Rhizobium alkalisoli CCBAU 01393 (EU074168) 77 Rhizobium vignae CCBAU 05176 T(GU128881) Rhizobium pseudoryzae J3-A127 T(DQ454123) Bradyrhizobium japonicum USDA 6 T (NC017249)
0.01 Figure 26: Neighbour-joining tree based on nearly complete 16S rRNA gene sequences of strain BN-19 T ( Rhizobium pakistanensis sp. nov.) and the recognized species of genus Rhizobium.
139 146
T 92 R. yanglingense SH 22623 (AY907373) 99 R. gallicum R602sp T (AY907371) 99 R. mongolense USDA 1844 T (AY907372) R. sullae IS123 T (DQ345069) R. lusitanum P1-7T (DQ431671) T 53 R. mesosinicum CCBAU 25010 (EU120726) R. etli USDA 9032 T (AJ294404) R. multihospitium CCBAU 83401 T (EF490019) 98 R. tropici IIB USDA 9030 T (AM418789) 66 R. fabae CCBAU 33202 T (EF579929) 97 R. leguminosarum USDA 2370 T (AJ294405) R. soli DS-42 T (GQ260191) 63 R. giardinii R4385 T (AM418780) T R. tarimense PL-41 (JF508524) R. cellulosilyticum LMG 23643 T (AM286429) R. pakistanensis BN-19 T (AB856324) 66 63 R. huautlense SO2 T (AM418782) R. alkalisoli CCBAU 01393 T (EU672461) 79 R. galegae USDA 4128 T (AM418779)
0.01
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.
140 147
98 R. yanglingense SH 22623 T (AY907359) 100 R. gallicum R602sp T (AY907357) R. mongolense USDA 1844 T (AY907358) R. alkalisoli CCBAU 01393 T (EU672490) 95 R. huautlense SO2 T (AM182128) R. giardinii R4385 T (AM182123) T R. galegae USDA 4128 (AM182127) R. soli DS-42 T (GQ260192) R. lusitanum P1-7T (DQ431674) T R. cellulosilyticum LMG 23642 (AM286427) R.tropici IIA USDA_9039 T (AJ294372) T R. multihospitium CCBAU 83401 (EF490029) 69 R. tropici IIB USDA 9030 T (AJ294373) R. mesosinicum CCBAU 25010 T (EU120732) R. etli CFN 42 T (CP000133) R. leguminosarum USDA 2370 T (AJ294376) T 91 R. fabae CCBAU 33202 (EF579941) 97 R. pisi DSM 30132 T (EF113134) R. pakistanensis BN-19 T (AB855792) R. tarimense PL -41 T (JF508523)
0.02
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.
141 148
T 85 R. multihospitium CCBAU 83401 (EF490040) 76 R. tropici IIB USDA 9030 T (AF169584) 69 R. tropici IIA USDA 9039 T (AF169583) T R. lusitanum P1-7 (EF639841) R. galegae USDA 4128 T (AF169587) T R. etli CFN 42 (AF169585) 54 R. leguminosarum . USDA 2370 T (EU155089) T R. tibeticum CCBAU 85039 (EU407190) R. alkalisoli CCBAU 01393 T (EU672475) R. pakistanensis BN-19 T (AB856325) T R. gallicum USDA 3736 (AY929427) 100 R. mongolense USDA 1844 T (AY929453) S. xinjiangense CCBAU 110 T (EU155088)
0.02
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.
142 149
Table 22: Sequence similarities (%) for 16S rRNA gene, atpD , recA , glnII and DNA-DNA relatedness between
Rhizobiumpakistanensis BN-19 T and reference strains.
Strain BN-19 T
16S atpD recA glnII DNA-DNA relatedness rRNA R. alkalisoli CCBAU 01393 T 97.4 93.3 83.5 89.0 20.56
R. tarimense PL-41 T 97.3 89.5 80.6 - 20.49
R. vignae CCBAU 05176 T 97.3 91.2 85.7 88.8 22.51
R. huautlense SO2 T 97.2 93.3 85.4 - 15.91
R. leguminosarum USDA 2370 T 96.4 85.8 85.8 86.7 15.58
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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-19 T and reference strains are described in Table 23. Thirteen different types of fatty acids were
T detected in BN-19 , of which C 19:0 cyclo ω8c (22.52%), summed feature 2 (C 14:0 3-
OH and/or C 16:1 iso 1; 30.37) and summed feature 8 (C 18:1 ω7c; 24.07) were the major fatty acids. Some fatty acids like C 16:0 , C 19:0 cyclo ω8c, summed feature 2, 3 and 8 were found in all tested strains.
Based on all the results obtained, strain BN-19 T 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-19 T (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).
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Table 26: Cellular fatty acid contents (%) of strain BN-19 T 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 - C17:1 ω8c - - - 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 C18:1 ω9c - - - - 0.81 C18:1 ω7c 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 C20:1 ω7c - - - 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-19 T; 2 R. alkalisoli CCBAU 01393 T; 3 R. tarimense PL-41 T; 4 R. vignae CCBAU 05176 T; 5 R. huautlense SO2 T; 6 R. leguminosarum USDA 2370 T.
Summed features are groups of two or three fatty acids that cannot be separated by GLC using the MIDI system. Summed feature 1 comprises C 15:1 iso H and/or C 13:0 3-OH, summed feature 2 comprises C 14:0 3OH/C 16:1 iso 1, summed feature 3 comprises C 16:1 ω7c and/or C 16:1 ω6c, summed feature 8 comprises C 18:1 ω6c.
(-) 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 N 2-fixation was recorded and result revealed that all selected PGPR strains significantly increased nodule number, pod yield, biomass yield and N 2-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 N 2-fixation. Crop parameters studied
-1 -1 include biomass yield (t ha ), pod yield (t ha ), shoot N contents (%) and N 2-
15 fixation. N 2-fixation of groundnut was assessed by δ N 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-
19 T was isolated from groundnut nodules and validated by adopting minimal standards. The 16S rRNA gene sequence similarities of BN-19 T with closely related type strains R. alkalisoli CCBAU 01393 T, R. tarimense PL-41 T, R. vignae CCBAU
05176 T and R.huautlense SO2 T 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-19 T 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
T of the strain BN-19 were C19:0 cyclo ω8c, summed feature 2 (C 14:0 3-OH and/or
C16:1 iso 1) and summed feature 8 (C 18:1 ω7c). On the basis of the phylogenetic, physiological and phenotypic analyses, the strain BN-19 T 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-19 T (=LMG 27895 T=CCBAU 101086 T).
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.
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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%
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Appendix 2:CCBAU Genbank depoist certificate of BN-19 T
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Appendix 3:LMGGenbank depoist certificate of BN-19 T
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