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Understanding the Composition and Role of the Prokaryotic Diversity in the Potato Rhizosphere for Crop Improvement in the Andes

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Understanding the Composition and Role of the Prokaryotic Diversity in the Potato Rhizosphere for Crop Improvement in the Andes Understanding the composition and role of the prokaryotic diversity in the potato rhizosphere for crop improvement in the Andes Jonas Ghyselinck Dissertation submitted in fulfilment of the requirements for the degree of Doctor (Ph.D.) in Sciences, Biotechnology Promotor - Prof. Dr. Paul De Vos Co-promotor - Dr. Kim Heylen Ghyselinck Jonas – Understanding the composition and role of the prokaryotic diversity in the potato rhizosphere for crop improvement in the Andes Copyright ©2013 Ghyselinck Jonas ISBN-number: 978-94-6197-119-7 No part of this thesis protected by its copyright notice may be reproduced or utilized in any form, or by any means, electronic or mechanical, including photocopying, recording or by any information storage or retrieval system without written permission of the author and promotors. Printed by University Press | www.universitypress.be Ph.D. thesis, Faculty of Sciences, Ghent University, Ghent, Belgium. This Ph.D. work was financially supported by European Community's Seventh Framework Programme FP7/2007-2013 under grant agreement N° 227522 Publicly defended in Ghent, Belgium, May 28th 2013 EXAMINATION COMMITTEE Prof. Dr. Savvas Savvides (chairman) Faculty of Sciences Ghent University, Belgium Prof. Dr. Paul De Vos (promotor) Faculty of Sciences Ghent University, Belgium Dr. Kim Heylen (co-promotor) Faculty of Sciences Ghent University, Belgium Prof. Dr. Anne Willems Faculty of Sciences Ghent University, Belgium Prof. Dr. Peter Dawyndt Faculty of Sciences Ghent University, Belgium Prof. Dr. Stéphane Declerck Faculty of Biological, Agricultural and Environmental Engineering Université catholique de Louvain, Louvain-la-Neuve, Belgium Dr. Angela Sessitsch Department of Health and Environment, Bioresources Unit AIT Austrian Institute of Technology GmbH, Tulln, Austria Dr. Barbara Doyle Prestwich School of Biological, Earth and Environmental Sciences University College Cork, Cork, Ireland iii DANKWOORD Doctoreren omvat het zelfstandig uitdenken en uitvoeren van experimenten, het schrijven van publicaties (meestal gevolgd door het schrijven van een rebuttal), het halen van de deadline voor het indienen van een doctoraatsthesis en de uiteindelijke verdediging ervan. Dit alles zou uiteraard niet mogelijk geweest zijn zonder de hulp van vele mensen. Vooreerst is er mijn promotor Prof. Paul De Vos. Paul, u heeft me de vrijheid gegeven om telkens de richting in te slaan die ik wilde uitgaan met mijn onderzoek, zonder veel op mijn vingers te kijken. Op geregelde tijdstippen vroeg u hoe de huidige stand van zaken was; de daaruit voortvloeiende reactie maakte wel duidelijk of ik al dan niet in de goede richting aan het gaan was. Ondanks het feit dat die vrijheid mij toeliet onderzoek te doen in wat mij echt interesseerde, werd op die manier ook een zekere zelfstandigheid en verantwoordelijkheid opgelegd; iets waar al uw doctoraatstudenten ongetwijfeld sterk uitkomen. Paul, bedankt dat ik onder uw toezicht aan een doctoraat mocht beginnen, bedankt voor het geven van feedback wanneer nodig, voor het nalezen van geschreven papers en het uiteindelijke doctoraat, en niet te vergeten voor de lekkere truffels van de volley die regelmatig in het koffiekot terug waren te vinden! Een aantal maanden na de start van mijn doctoraat werd een nieuwe post-doc aangenomen. Dr. Kim Heylen kwam op het juiste moment om mijn onderzoek bij te sturen in de richting van een eerste paper. Met jouw ervaring en kennis binnen het domein van de microbiologie was het mogelijk om tegenwerk te bieden waar nodig; iets wat resulteerde in stimulerende en richtinggevende discussies. Kim, dank je wel voor de vele discussies bij de opzet van nieuwe experimenten, voor het grondig nalezen van meerdere tussenversies van papers en voor de leuke babbels over reizen, films en Deense reeksen in het koffiekot. Bedankt voor je grote bijdrage aan dit doctoraat; je rol als begeleider heb je uitstekend ingevuld! I would like to thank the people with whom I cooperated frequently, and whom I met for the first time at our annual meeting in Lima, Peru. Dr. Angela Sessitsch, Dr. Barbara Doyle Prestwich, Stefan Pfeiffer and Siva Velivelli, thank you very much for sharing results and expertise which made joint publication possible and which also allowed me to gain new insights within the broad field of microbiology. You were not only excellent researchers to work with, but also very pleasant people to travel with! I would also like to thank all the people that were part of the Jury of my PhD defense (Prof. Paul De Vos, Dr. Kim v Heylen, Prof. Stéphane Declerck, Prof. Savvas Savvides, Prof. Anne Willems, Dr. Angela Sessitsch, Dr. Barbara Doyle Prestwich and Prof. Peter Dawyndt) for carefully proofreading my PhD thesis, providing comments that led to improvement of the manuscript and of course for allowing me to defend my PhD. Uiteraard wil ik mijn collega’s van de PDV groep bedanken voor de zeer leuke werksfeer; Ines, Bram, Joanna en – door velen aanzien als mijn tweelingbroer – Sven. Ik kon mij geen betere collega’s indenken! De vele frietkotbezoeken op vrijdagmiddag, de etentjes aan de vijver bij mooi weer, de lachmomenten en de koffiepauzes in het koffiekot ga ik niet snel vergeten. Ook de meeting ter ere van de 100-jarige verjaardag van NvvM in Nederland is er één om niet snel te vergeten. Kort gezegd; ’t was wijs met jullie, ik heb mij enorm geamuseerd! My colleague PhD student and good friend Stefan Pfeiffer; thank you, not only for the excellent cooperation we had during our joint research but also for the pleasant (but often intense) travelling we did during the weeks following annual meetings. I must say that during our trips in Peru and Bolivia, together with our companion Prof. Arthur Schüssler, we probably had several of the best laughs ever; one of them including Stefan’s struggle with the word ‘Ollantaytambo’ and the fact that some people tend to ‘eat coffee’. I’m really happy to have met you guys during the VALORAM project; and to have travelled with you from the tropical regions near the Amason basin to the isolated high altitudes of the Altiplano of South-America. This was an adventure! I sincerely hope we meet again in the very near future! Door de input van vele collega’s is dit werk uiteindelijk geworden tot wat het is. Bedankt Liesbeth, Emly en Evi voor de vele sequenties die jullie hebben gelopen; vooral tijdens de laatste maanden van mijn doctoraat. Liesbeth, bedankt voor de hulp in het labo, en voor de grappige momenten. An Coorevits, heel erg bedankt voor de uren uitleg wat betreft de complexe taxonomie van Pseudomonas; zelfs in de periode dat je zelf druk bezig was met het schrijven van jouw doctoraat. Bedankt voor de uitleg rond ARB, en de bijdrage tot de laatste paper! Isabel, de discussies gaande van het uitdenken van pyrosequencing experimenten tot hoe interessant het eigenlijk wel niet is om een huis te kopen en te verbouwen hebben mij nieuwe inzichten gegeven. Timo en Anne Becker, met jullie was het steeds leuk om muziekervaringen te delen, gaande van persoonlijke dj-sets, tot de concerten van Anne en de optredens van Timo (waarvoor ik mijn schade nog moet inhalen). Koenraad, heel erg bedankt voor je toewijding en het uitdenken van de statistiek wat betreft de eerste paper. Wim De Smet en Bart Verheyde, bedankt voor het schrijven van scripts die mijn werk aanzienlijk sneller deden gaan. Bart Hoste en Margo, heel erg bedankt voor de weelderige uitleg als er ergens problemen of vi onduidelijkheden waren. Nadine en Claudine, bedankt voor het verzenden van stammen en het verwerken van de vele permits die daarmee gepaard gingen. Mijn ouders en zus! Zonder jullie was ik nooit aan een doctoraat begonnen; doctoreren stond ver van mijn bed. Dankzij jullie motiverende en stimulerende woorden ben ik er uiteindelijk toch aan begonnen en ik moet zeggen, het was de juiste beslissing; een 23-jarige weet blijkbaar toch niet alles beter. Ook jullie interesse in mijn werk heb ik enorm geapprecieerd. Bedankt voor het aanbieden van een warm nest tijdens de vier jaren van m’n doctoraat, maar ook voor de vele jaren daarvoor en ongetwijfeld voor de vele jaren die nog moeten komen! Mijn vriendin Julie! Doorheen de vele jaren dat we elkaar kennen heb ik me altijd gesteund gevoeld in mijn werk en in het feit dat ik mijn doctoraat zou behalen. Je hebt me bijgestaan waar nodig; het laatste jaar was druk en ik was in de laatste maanden misschien niet altijd de meest attente partner, maar toch hebben we het samen heel goed doorgemaakt. Heel binnenkort is het tijd voor de vakantie waar we zo lang naar hebben uitgekeken. We gaan het er eens goed van pakken! Samen op naar al het nieuws dat nog komen zal! vii LIST OF ABBREVIATIONS AA Amino Acid ACC 1-aminocyclopropane-1-carboxylate AG Anastomosis Group AMF Arbuscular Mycorrhizal Fungi ANI Average Nucleotide Identity CHCA cyano-4-hydroxycinnamic acid dNMS diluted Nitrate Mineral Salts FAME Fatty Acid Methyl Ester FAO Food and Agriculture Organization IAA Indola Acetic Acid ISR Induced Systemic Resistance LGT Lateral Gene Transfer LTP Living Tree Project MALDI-TOF Matrix Assisted Laser Desorption Ionisation Time Of Flight MS Mass Spectrometry MDS Multi Dimensional Scaling ML Maximum Likelihood MLSA Multi Locus Sequence Analysis MPCA Microbial Pest Control Agent MSDS Material Safety Data Sheet NBRIP National Botanical Research Institute’s Phosphate NFL Nearly Full-Length NGS Next Generation Sequencing OECD Organization for Economic Co-operation and Development OTU Operational Taxonomic Unit PBS Phosphate Buffered Saline
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