Interactions between non-symbiotic N_1tn2-fixing bacteria and plant roots in plant-microbial associations [Elektronische Ressource] / presented by Olga-Cristina Calvo Alegre

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Extrait

Informations

Publié par	universitat_hohenheim
Publié le	01 janvier 2009
Nombre de lectures	34
Langue	English
Poids de l'ouvrage	3 Mo

Extrait

Institute of Plant Nutrition
University of Hohenheim
Prof. Dr. Nicolaus von Wirén

Interactions between non-symbiotic N -fixing bacteria 2
and plant roots in plant-microbial associations

Dissertation

Submitted in fulfilment of the requirements for the degree
―Doktor der Agrarwissenschaften―
(Dr.sc.agr. / Ph.D. in Agricultural Sciences)

to the
Faculty of Agricultural Sciences

presented by

Olga-Cristina Calvo Alegre
Cornellà de Llobregat
(Catalonia)
2009

This thesis was accepted as a doctoral dissertation in fulfilment of the
requirements for the degree ―Doktor der Agrarwissenschaften― by the Faculty
thAgricultural Sciences at University of Hohenheim on 3 March 2009.

thDate of oral examination: 23 October 2009

Examination Committee
Supervisor and Review Prof. Dr. Nicolaus von Wirén
Co-Reviewer PD Dr. Joseph Strauss
Additional examiner Prof. Dr. Georg Cadisch
Vice-Dean and Head of the Committee Prof. Dr. Werner Bessei

Aquest treball està dedicat
a l´Anna, al David i a
tota la meva família
Table of contents

1 SUMMARY - ZUSAMMENFASSUNG
1.1 Summary ........................................................................................................ 1
1.2 Zusammenfassung ......................................................................................... 3
2 INTRODUCTION
2.1 Nitrogen nutrition ............................ 5
2.1.1 Forms of nitrogen in agricultural ecosystems ........................................... 5
2.1.2 Ammonium uptake, assimilation and transport system in plants .............. 7
2.1.3 Biological N fixation (BNF) .................................... 11 2
2.1.3.1 Symbiotic N fixation ................................... 12 2
2.1.3.2 Non-symbiotic N fixation ............................ 13 2
2.1.3.3 Environmental factors influencing non-symbiotic N fixation ....... 16 2
2.1.3.4 Methods to quantify biological N fixation ................................... 17 2
2.2 Plant growth promoting effects of diazotrophs independent of N 2
fixation .......................................................................... 20
2.2.1 Production of plant growth promoting substances ................................. 20
2.2.1.1 Phytohormones .......... 20
2.2.1.2 Enzymes ..................................................................................... 23
2.2.1.3 Production of vitamins ................................ 24
2.2.2 Interaction with other microorganisms .................... 24
2.2.2.1 Antagonism (Biocontrol) ............................................................. 24
2.2.2.2 Synergism................................................... 25
2.2.3 Enhanced mobilization of nutrient elements ........................................... 26
2.2.4 Enhanced stress tolerance ..................................... 28
2.2.5 Associative interactions between plant roots and diazotrophic
bacteria .................................................................. 29
2.2.5.1 Plant signalling compounds recognized by microbes ................. 30
2.2.5.2 Microbial signalling compounds recognized by the plants .......... 34
2.2.5.3 Bacterial motility ......................................................................... 34
2.2.5.4 Role of bacterial exopolysaccharides in root colonization .......... 35
2.3 Objective and research questions 37 3 MATERIAL AND METHODS
3.1 Inoculation experiments in the greenhouse .................................................. 38
3.1.1 Preparation of soil ................................ 38
3.1.2 Plant material and growth conditions ..................... 39
3.1.3 Preparation of bacterial inoculants and inoculation method ................... 42
3.1.4 Analysis of growth and yield measures .................................................. 44
3.2 Plant root exudates ...................................................... 45
3.2.1 Collection of seed and root exudates ..................... 45
3.2.2 Characterization of exudates by HPLC analysis .................................... 47
3.3 Chemotaxis assays ...................................................................................... 49
3.3.1 Bacteria strains and media ..................................... 49
3.3.2 Motility assay .......................... 49
3.3.3 Detection of bacterial exopolysaccharides (EPS) by Calcofluor
staining …………………… ..................................................................... 52
3.4 Hydroponic culture of tomato ........ 52
3.4.1 Hydroponic culture conditions 52
3.4.2 Physiological analysis ............................................................................ 53
153.4.3 N-ammonium uptake studies ............................... 55
3.5 Plant molecular biology ................ 55
3.5.1 Transformation of tomato plants (Lycopersicon esculentum L.) ............. 55
3.5.2 RNA gel blot analysis ................................................................ 56
4 RESULTS
4.1 Influence of different factors on growth of wheat plants
inoculated with diazotrophic bacteria ............................................................ 58
4.1.1 Nitrogen fertilization….………. ............................... 58
4.1.2 Soil type …………………… . .................................. 75
4.1.3 Plant genotype………….……….... ......................................................... 75
4.1.4 Bacteria strain….……………. . 77
4.1.5 Concentration of inoculum… .................................. 87
4.1.6 Inoculum delivery mode……. ................................. 87
4.2 Seed / root exudates analysis…. .. 88
4.3 Assays for bacterial motility and EPS production ......................................... 90
4.3.1 Motility in the presence of several pure substances ............................... 92
4.3.2 Motility in the presence of seed / root exudates ..... 93 4.3.3 Bacterial macrocolony patterns .............................................................. 96
4.4 Physiological characterization of LeAMT1;1 and LeAMT1;2 in tomato ......... 99
4.4.1 Characterization of the contribution of LeAMT1;1 to ammonium
uptake and the N nutritional status of tomato ........................................ 99
4.4.2 Characterization of the contribution of LeAMT1;2 to ammonium
uptake and the N nutritional status of tomato plants ........................... 110
5 DISCUSSION
5.1 Contribution of non-symbiotic rhizobacteria to nitrogen nutrition ................ 120
5.2 Factors influencing plant growth promotion by rhizobacteria ...................... 124
5.3 Bacterial responses to root exudates ......................................................... 135
5.3.1 Effect of exudates on surface migration ............... 136
5.3.2 Effect of exopolysaccharides production on surface motility ................ 140
5.4 Possible contribution of LeAMT1;1 and LeAMT1;2 to nitrogen
nutrition in tomato plants ............................................................................. 144
6 REFERENCES ...................................... 149
7 APPENDIX ............ 173
8 ACKNOWLEDGMENTS ........................................................................................ 176
9 CURRICULUM VITAE ........................... 178 LIST OF ABBREVIATIONS

ABA abscisic acid
ACC 1-aminocyclopropane-1-carboxylic acid
ADP adenosine diphosphate
AMT ammonium transporter
ARA acetylene reduction assay
ATP adenosine 5’-triphosphate
BNF biological nitrogen fixation
CFU colony forming units
CPS capsular polysaccharides
cv. cultivar
d days
DNA deoxyribonucleic acid
DW dry weight
EPS exopolysaccharides
EtBr ethidium bromide
GA gibberellic acid
HATS high-affinity transport system
HMW high-molecular-weight
HPLC high performance liquid chromatography
IAA indole-3-acetic acid
IBA indole-3-butyric acid
ISR induced systemic resistance
LATS low-affinity transport system
LMW low-molecular-weight
LPS lipopolysaccharides
Napp apparent nitrogen mineralization
MS Murashige-Skoog medium
Nfert fertilized nitrogen
Nmin mineral nitrogen
NR nitrate reductase
OD optical density at 600 nm 600
OPA ortho-phthalaldehyde buffer
ORF open reading frame
PBS phosphate buffer
PCR polymerase chain reaction
PGPR plant growth promoting rhizobacteria
PHB poly-ß-hxdroxybutyrate
RH relative humidity
RNA ribonucleic acid
R/S root to shoot ratio
RT room temperature
SD standard deviation
SE standard error
sp. species (singular)
SPAD spectral plant analysis diagnostic
spp. species (plural)
TE Tris-EDTA buffer
UV ultraviolet
vs. versus
v/v volume per volume
WHC water holding capacity
WT wild typeSummary – Zusammenfassung
1. SUMMARY – ZUSAMMENFASSUNG

1.1. Summary

The development of biofertilizers on the basis of plant growth promoting rhizobacteria
(PGPR) may be a promising approach to