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Improvement of common bean (Phaseolus vulgaris) nodulation by selected rhizobial strain from Egyptian soils through genotypic characterization, symbiotic effectiveness and competitiveness under salt stress conditions [Elektronische Ressource] / vorgelegt von Abdelaal Ali Yousef Shamseldin

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Improvement of common bean (Phaseolus vulgaris) nodulation by selected rhizobial strains from Egyptian soils through genotypic characterization, symbiotic effectiveness and competitiveness under salt stress conditions Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) dem Fachbereich Biologie der Philipps-Universität Marburg/Lahn vorgelegt von Abdelaal Ali Yousef Shamseldin aus Menoufia, Ägypten Marburg/Lahn 2005 Vom Fachbereich Biologie der Philipps-Universität Marburg Als Dissertation angenommen am.....................................2005 Erstgutachter Prof. Dr. Dietrich Werner Zweitgutachter Prof. Dr. Erhard Mörschel Tag der mündlichen Prüfung am.......................................2005 Dedication To my whole family who have given me much love and support The following parts of this thesis have been published in: Shamseldin, A. and Werner, D. 2004. Selection of competitive strains of Rhizobium nodulating Phaseolus vulgaris and adapted to environmental conditions in Egypt, using the gus-reporter gene technique. World Journal of Microbiology and Biotechnology, 20:377-382. Shamseldin, A., Vinuesa, P., Thierfelder, Heidemarie and Werner, D. 2005.

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Published 01 January 2005
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Improvement of common bean (Phaseolus vulgaris) nodulation by
selected rhizobial strains from Egyptian soils through genotypic
characterization, symbiotic effectiveness and competitiveness
under salt stress conditions






Dissertation
zur
Erlangung des Doktorgrades
der Naturwissenschaften
(Dr. rer. nat.)



dem
Fachbereich Biologie
der Philipps-Universität Marburg/Lahn

vorgelegt von


Abdelaal Ali Yousef Shamseldin

aus Menoufia, Ägypten





Marburg/Lahn
2005
































Vom Fachbereich Biologie der Philipps-Universität Marburg
Als Dissertation angenommen am.....................................2005

Erstgutachter Prof. Dr. Dietrich Werner
Zweitgutachter Prof. Dr. Erhard Mörschel

Tag der mündlichen Prüfung am.......................................2005








Dedication









To my whole family who have given
me much love and support



















The following parts of this thesis have been published in:


Shamseldin, A. and Werner, D. 2004. Selection of competitive strains
of Rhizobium nodulating Phaseolus vulgaris and adapted to
environmental conditions in Egypt, using the gus-reporter gene
technique. World Journal of Microbiology and Biotechnology,
20:377-382.

Shamseldin, A., Vinuesa, P., Thierfelder, Heidemarie and Werner, D.
2005. Rhizobium etli and Rhizobium gallicum nodulate Phaseolus
vulgaris in Egyptian soils and display cultivar-dependent symbiotic
efficiency. Symbiosis, 38:145-161.

Shamseldin, A. and Werner, D. 2005. High salt and high pH tolerance
of new isolated Rhizobium etli strains from Egyptian soils. Current
Microbiology, In press.

Publication submitted:

Shamseldin, A., Naylwidhe, J. and Werner, D. 2005. A proteomic
approach towards the analysis of salt tolerance in Rhizobium etli and
Sinorhizobium meliloti strains.

Reprints at the end of the thesis.
i
List of abbreviations


AMPD 2-amino-2-methyl-1,3 propanediol
APS ammonium peroxide persulfate
ARA acetylene reduction assay
ARDRA amplified ribosomal DNA restriction analysis
bp base pair
BLAST basic local alignment search tool
bv biovar
CBB commassie brilliant blue
CHAPS [3-cholamidopropyl-dimethyl amonio ]-1-propane sulfonate
α CHCA α-cyano-4-hydroxycinnamic acid
CTAB cetyltrimethyl ammonium bromide
DMF dimethyl formamide
DMSO sulfoxide
DNA deoxyribonucleic acid
DIG digoxigenin
dt dry ton
dNTP’s neuclotide bases
DTT dithiothreitol
EC electric conductivity
EDTA etylenedriamintetra acetic acid
ERIC repetitive intergenic consensus
EtBr ethidium bromide
Et ethanol
FAOSTAT Food Agricultural Organization
fix symbiotic nitrogen fixation gene
FW fresh weight
g gram
h hour
ha hectare
HOMOPIPES hexamethyleneimine
IEF isoelectric focusing
IPG immobiline pH gradient
ITS internally transcribed spacer
Kb Kilobase
KD Kilodalton
LB Lauria Bertani, a medium for E. coli
LSD least significant difference
M molarity
mA milliamper
MALDI matrix assisted laser adsorption ionization ii
mbar millibar
Mb mega base pair(s)
MD mega Dalton
MES morpholinoethanesulphonic acid
mg milligram
min minute
MM minimal media
MS mass spectrometer
MW molecular weight
NBT nitro blue tetrazolium salt
NCBI National Center for Bioinformatics Institute
nif nitrogenase structural gene
nm nanometer
nod nodulation genes
OD optical density
PAGE polyacrylamide gel electrophoresis
PCR polymerase chain reaction
+pH reciprocal of the H concentration
PMF peptide mass fingerprint
ppm parts per million
PY tryptone yeast
rDNA ribosomal DNA
REP repetitive extragenic palindromic sequences
RFLP restriction fragment length polymorphism
RNA ribonucleic acid
rpm revolutions per minute
rt room temperature
SDS sodiumdodeocyl sulphate
sec second
Str streptomycin
Sp spectinomycin
TAE tris acetate EDTA
TBE tris borate EDTA
TCA trichloroacetic acid
TE tris EDTA
TEMED N, N, N, N, tetramethylethylenediamine
TFA trifluro acetic
TIEF tagged image file formate
TOF time of flight
tRNA transfer ribonucleic acid
UPGMA Unpaired group method using arithmetic averages
UV ultra violet
V volt iii
v/cm volt per centimeter
v/v volume by volume
W watt
Wt wild type
w/v weight per volume
x-gluc 5-bromo-4-chloro-3-indoalyl glucuronide
x-phosphate o-4-chloro-3-indoalyl phosphate salt




































iv
Table of contents
List of abbreviations
1 Introduction 1
1.1 General introduction
1.2 Taxonomy of Rhizobium nodulating Phaseolus vulgaris 2
1.3 Differentiation among rhizobial isolates using molecular methods 4
1.3.1 PCR based on the repetitive extragenic palindromic (REP) 4
sequences
1.3.2 Characterization of rhizobial isolates using Amplified Ribosomal 5
DNA Restriction analysis (ARDRA)
1.3.3 Typing of rhizobial isolates according to their plasmid profiles 6
1.3.4 Identification of rhizobial isolates using 16S rDNA sequencing and 7
phylogenetic tree
1.3.5 Hybridization with nifH gene 8
1.4 Factors that limit the response of rhizobial inoculation in the field 8
1.4.1 Biotic factors and competition between indigenous and inoculant 8
rhizobial strains
1.4.2 A biotic factors: 9
1-salinity of soil 9
2- Soil pH 10
3- Soil temperature 11
4- Availability of nitrogen 12
1.5 Genes involved in salt tolerance 12
2 Materials and Methods 14
2.1 Isolation of rhizobia from bean nodules and standard strains 14
2.2 Isolation of genomic DNA from rhizobial strains 15
2.3 Estimation of DNA concentration 16
2.4 Methods of molecular characterization 17
2.4.1 REP-PCR genomic fingerprinting 17
2.4.2 Amplified ribosomal DNA restriction analysis (ARDRA) 17
2.4.3 Analysis of REP-PCR fingerprints and rDNA restriction patterns 20
2.4.4 Amplification of nodC genes 20
2.4.5 Plasmid DNA content 20
2.4.6 Direct sequencing of 16S rDNA 21
a- Preparation of sequencing gel 21
b- Comparing the sequence analysis based on computer programs 22
c- 16S rDNA sequence alignment and phylogeny estimation 22
2.4.7 Transfer of DNA from agarose gels to nylon membranes (southern 22
blot)
2.4.8 Generation of digoxigenin-labelled nifH-DNA probe 23
2.4.9 Southern hybridization with DIG-labelled DNA probe and signal 24
detection
2.5 Nodulation experiments 26
2.5.1 Cultivars of common bean (Phaseolus vulgaris) v
2.5.2 Seeds germination and sterilization 26
2.5.3 Plant nodulation assays 26
2.5.4 Estimation of the acetylene reduction assay (ARA) 27
2.5.5 Physiological trials to select strains adapted to environmental 28
conditions
2.5.6 Plant nodulation assays and symbiotic performance under stress of 28
salinity or alkalinity
2.6 Competition experiments 29
+2.6.1 Construction of a R. tropici CIAT 899 Gus strain
2.6.2 Competition under normal conditions or stress of salinity, or 30
alkalinity
2.6.3 Gus staining 31
2.7 Effect of nitrogen fertilizer on the symbiotic efficiency of R. etli 31
strains to nodulate Phaseolus vulgaris
2.8 Proteom analysis 32
2.8.1 Protein isolation and sample preparation
2.8.2 Preparation of samples for 2D gels electrophoresis 32
2.8.3 Running of first and second dimension 32
2.8.4 Preparation of 2-D gels 33
2.8.5 Handling of protein spots for analysis by MALDI-TOF 33
2.8.6 Labelling of protein with Cyanine dyes 35
3 Results 36
3.1 Efficiency and host specificity of rhizobial strains 36
3.2 Genotypic characterization of bean rhizobial isolates from Egyptian 41
soils using different finger printing methods
3.2.1 Typing of bean rhizobial isolates by 16S rDNA-PCR/RFLP analysis 42
3.2.2 ates by 23S rDNA-PC 45
3.2.3 Plasmid profile analysis for rhizobial strains 48
3.2.4 Typing of bean rhizobial isolates by ITS rDNA-PCR/RFLP analysis 51
3.2.5 16S rDNA sequencing and identification of rhizobial isolates 52
3.2.6 Combination between the RFLP analysis of 16S rDNA and 23S 57
rDNA
3.2.7 Phylogenetic analysis of rrs sequences 58
3.2.8 Hybridization with nifH gene 59
3.3 Phenotypic characteristics of rhizobial strains 61
3.4 Physiological characteristics of strains to select the candidates 63
adapted to environmental stresses
3.5 Selection of competitive strains of Rhizobium nodulating Phaseolus 70
vulgaris and adapted to soil stress conditions in Egypt, using Gus-
reporter gene technique
3.6 Effect of nitrogen fertilizer application on nodulation of Phaseolus 78
vulgaris
3.7 Proteom analysis of salt tolerance in R. etli strain EBRI 26 80
3.8 Protein expression in Sinorhizobium meliloti strain 2011 after salt 83 vi
stress
4 Discussion 88
4.1 Genetic biodiversity of bean rhizobial isolates and identification
4.2 Efficiency and compatibility between plant host and rhizobial strains 91
4.3 Selection of well adapted strains to environmental stresses 92
4.4 Symbiotic performance of rhizobial strains under high salt or high 93
pH
4.5 Selection of the most competitive and well adapted strains to 94
environmental conditions in Egypt using Gus-reporter gene
4.5.1 Marking of a R. tropici CIAT 899G with the Gus gene 94
4.5.2 Competition between strain CIAT 899 wt and its derivative CIAT 94
899G
4.5.3 Selection of competitive strains under normal conditions 94
4.5.4 of competitive strains under salt stress 95
4.5.5 Selection of competitive strains under alkalinity stress 96
4.5.6 Selection of competitive strains under temperature stress 96
4.6 Effect of nitrogen fertilizer application and rhizobial inoculation on 97
nodulation of Phaseolus vulgaris
4.7 Approaches of proteom analysis of R. etli strain EBRI 26 under salt 97
stress
4.7.1 CBB staining and MALDI-TOF analysis 97
4.7.2 Second approach of CyDyes labelling 98
4.8 A proteomic approach of Sinorhizobium meliloti strain 2011 under 99
salt stress
5 Summary 102
6 Zusammenfassung 108
7 References 115
Appendix 137
Acknowledgements 150