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Introduction production of siderophores (Schwyn & Neilands, 1987). Comparison of both Pss22d and Psg1a on this medium shows remarkably different phenotype of the strains (Fig.5A). The antagonist produces a much larger halo as compared to the pathogen. The same tendency was observed in several low-iron liquid media after quantification of siderophore activity in the supernatants (Fig.5B; Büttner, 2003). This difference is especially interesting, as the various pathovars of P. syringae are known to produce the same pyoverdin-type siderophore and so far there has been no experimental prove for the presence of other siderophores aside of this pyoverdin (Jülich et al., 2001). Siderophore production of Pss22d and its influence on the biocontrol system was addressed by the current thesis. I) II) DFOM-Equiv. [µM] 180 160 140 120 100 80 60 40 20 Pss22d/93 Psg1a/96 0 CAA KB Pipes 5b SM Medium Fig. 5A. Siderophore-production on CAS-agar. Bacterial syspension of I) Psg1a and II) Pss22d was applied to CAS-agar plate and incubated at 28°C for 48 h. Fig. 5B. Siderophore-production in different low-iron media after 48h of growth. Siderophore production of Pss22d ( ) and Psg1a ( ) was determined by CAS-assay and normalized to OD 600nm =1. Source of data: Büttner, 2003 17
Introduction 2.4 Aim of this study The antagonism of P. syringae pv. syringae 22d/93 (Pss22d) against bacterial blight of soybean caused by P. syringae pv. glycinea (Psg) has been successfully demonstrated in vitro, in planta, and under field conditions (May et al., 1997a; May et al., 1997b; Völksch et al., 1996; Völksch & May, 2001a). The outstanding performance of Pss22d accompanied by the multiple background information on the pathosystem sets this biological control apart from the large number of screenings for new control agents. It offers a unique opportunity to investigate active principle(s) of biological control in detail. First results implied a potential involvement of siderophore production (Fig.5). It was the aim of this study to investigate the influence of siderophore production on the biological control system by methods of molecular biology. A primary step for this was the determination of the siderophores produced by Pss22d. Mutational approaches such as marker-exchange mutagenesis and transposon mutagenesis were applied in order to identify the respective gene cluster responsible for siderophore biosynthesis. Pss22d and its siderophore mutants were analysed for their ability to grow under iron limited conditions in vitro. A detailed quantification of siderophores during in vitro growth was performed for a better comparison between siderophore production of Pss22d and Psg1a. The in planta application of Pss22d and the generated siderophore mutants was conducted to directly address the impact of siderophore production on biological control ability and epiphytic fitness of Pss22d. Finally, the distribution of a newly identified siderophore among different pathovars of P. syringae was analysed. 18
Page 1 and 2: Siderophore production of Pseudomon
Page 3 and 4: Acknowledgements This project was s
Page 5 and 6: Abbreviations Ach achromobactin-lik
Page 7 and 8: Contents 4.1.2 Pseudomonas syringae
Page 9 and 10: Contents 5.3 Influence of sideropho
Page 11 and 12: Introduction 2 Introduction Like hu
Page 13 and 14: Introduction In contrast to the rhi
Page 15 and 16: Introduction 2.1.2 Host, pathogen a
Page 17 and 18: Introduction The relationship betwe
Page 19 and 20: Introduction developement of resist
Page 21 and 22: Introduction Fe 2+ + H 2 O 2 → Fe
Page 23 and 24: Introduction 1997a; May et al., 199
Page 25: Introduction Pss22d demonstrated a
Page 29 and 30: Material and Methods Tab. 4. Antibi
Page 31 and 32: Material and Methods Iron-free 5b m
Page 33 and 34: Material and Methods Solution3, car
Page 35 and 36: Material and Methods 3.6 Plasmids T
Page 37 and 38: Material and Methods PCR-Screening
Page 39 and 40: Material and Methods on ice for 10
Page 41 and 42: Material and Methods stained in eth
Page 43 and 44: Material and Methods 4.2.5.3 Conver
Page 45 and 46: Material and Methods 4.2.5.9 Ligati
Page 47 and 48: Material and Methods strains were s
Page 49 and 50: Material and Methods 9.5). For sign
Page 51 and 52: Material and Methods gentle shaking
Page 53 and 54: Material and Methods columns were w
Page 55 and 56: Results 5 Results 5.1 Analysis of s
Page 57 and 58: Results The 13-kb open reading fram
Page 59 and 60: Results If integration is due to a
Page 61 and 62: Results Yersiniabactin is a siderop
Page 63 and 64: Results Next, a large number of tra
Page 65 and 66: Results Nevertheless the site of tr
Page 67 and 68: Results Achr2_KpnI_rev binding site
Page 69 and 70: Results Interestingly the mutant Ps
Page 71 and 72: Results XAD4-purification yielded i
Page 73 and 74: Results After changing the mobile p
Page 75 and 76: Results P P A Fig. 21. Isoelectic f
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Results reduced in comparison to th
Page 79 and 80:
Results A-E represents single inocu
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Results Tab. 10. Development of pop
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Results Tab. 11. Distribution of th
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Discussion bacteria with redundande
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Discussion 6.2 Possible regulation
Page 89 and 90:
Discussion stationary phase transit
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Discussion and bacterium (Loper et
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Literature Bultreys, A., Gheysen, I
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Literature Galperin, M. Y. (2006).
Page 97 and 98:
Literature Mazzola, M. (2002). Mech
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Literature Schubert, S., Rakin, A.,
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Literature Zou, J., Rodriguez-Zas,
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