Faculty of Sciences Department of Biochemistry and Microbiology Laboratory of Microbiology Diagnostics for quarantine plant pathogenic Clavibacter and relatives: molecular characterization, identification and epidemiological studies Joanna Załuga Promoter: Prof. Dr. Paul De Vos Co-promoter: Prof. Dr. Martine Maes Dissertation submitted in fulfillment of the requirements for the degree of Doctor (Ph.D.) in Sciences, Biotechnology Joanna Załuga – Diagnostics for quarantine plant pathogenic Clavibacter and relatives: molecular characterization, identification and epidemiological studies Copyright ©2013 Joanna Załuga ISBN-number: 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 promoters. 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 a QBOL (Quarantine Barcoding Of Life) project KBBE- 2008-1-4-01 nr 226482 funded under the Seventh Framework Program (FP 7) of the European Union. Publicly defended in Ghent, Belgium, October 11th, 2013 ii Examination Committee Prof. Dr. Savvas Savvides (Chairman) L-Probe: Laboratory for protein Biochemistry and Biomolecular Engineering Faculty of Sciences, Ghent University, Belgium Prof. Dr. Paul De Vos (Promoter) LM-UGent: Laboratory of Microbiology, Faculty of Science, Ghent University, Belgium BCCM-LMG Bacteria Collection, Ghent, Belgium Prof. Dr. Martine Maes (Co-promoter) Plant Sciences Unit – Crop Protection Institute for Agricultural and Fisheries Research (ILVO), Merelbeke, Belgium Dr. Peter Bonants Plant Research International (PRI) Business Unit Biointeractions & Plant Health, Wageningen, The Netherlands Ir. Johan Van Vaerenbergh Plant Sciences Unit – Crop Protection Institute for Agricultural and Fisheries Research (ILVO), Merelbeke, Belgium Dr. Pieter Stragier Laboratory for Microbiology Faculty of Sciences, Ghent University, Belgium Prof. Dr. Anne Willems Laboratory for Microbiology Faculty of Sciences, Ghent University, Belgium Prof. Dr. Monica Höfte Laboratory of Phytopathology, Department of Crop Protection Faculty of Bioscience Engineering, Ghent University, Belgium Prof. Dr. Peter Dawyndt Department of Applied Mathematics, Computer Science and Statistics PC-Lab of the Faculty of Science, Ghent University, Belgium iii Acknowledgements-Podziękowania To start I wish to thank prof. Dr. Paul De Vos who gave me the opportunity to carry out the PhD in the Laboratory of Microbiology. Your guidance, thoughtful insights and support were very important at every stage of my work. I am thankful that I could count on you, especially during the last few, very intensive months of my PhD. Your encouragement, advices and suggestions were invaluable for completing this work. I am grateful to the examination committee for their helpful suggestions, constructive remarks and fruitful discussions that helped me to improve my thesis. Prof. Dr. Martine Maes, thank you for your advice and comments on my publications and thesis. Dr. Peter Bonants, Prof. dr. Monica Höfte, Ir. Johan Van Vaerenbergh and dr. Pieter Stragier, thank you very much for taking your time to read my PhD and for your positive feedback. Kim, although you supervised me only at the beginning it was the most important time for me and I am thankful that I could work with you. Your wide knowledge and scientific input greatly influenced my first steps in the exciting scientific world. Writing the first article is never easy but with your guidance and support it was one of the most important and unforgettable experiences for me. I am grateful not only for your great scientific supervision and what you taught me during these four years but also for being a great colleague! Dear Johan, I would like to thank you very, very much for these four years. It was a great experience to work with you. I am grateful for showing me the real diagnostic lab, for all our discussions and for a wonderful time in ILVO. My dearest colleagues, Sven, Ines, Jonas and Bram, I spent with you the best time in the lab. You were the best company I could imagine to have during my PhD. Our coffee breaks discussions, Friday lunches, common conferences, meetings and practical exercises, I could not think of anyone better to share these great moments with. Sven, our philosopher and discussion man, thanks for all our chats and laughs and for being a super friend. Jonas, our potato/dj man, the most optimistic person I know, it was great to have someone so positive in the office. Ines, my best desk mate, thanks for your help at the beginning, for all the translations from Dutch, your support and the great moments we had together. Bram, the youngest in our team, hopping around, never tired and always full of ideas. Thanks for all the fun we had in the PDV group. Margo, you are an amazing person who has so much positive energy and who is always ready to help others. I will never forget your support and help, especially at the beginning of my adventure in LM-UGent and during my Erasmus exchange. Thank you for everything! Bart, Liesbeth, Joke, Evie, thanks for always answering my questions and for assistance in performing experiments in the lab. I would like to thank all colleagues from the Laboratory of Microbiology and from ILVO for creating a friendly atmosphere and for your enthusiastic support. It was a pleasure to work in such a nice environment. v I cannot forget my best friend Kamila who shared with me good and bad moments, who always was there when I needed her. Without you I would never be where I am now. I still remember our first days in Ghent when we started together. New places, new people, new challenges and even though it was tough we did it! Dzięki za wszystko moja najlepsza koleżanko! My friends, Kasie, Agi, Asie, Anie, Radek, Agata, Mati, Linda, Giacomo, Edyta, Marta, Lidka and many others I have always a wonderful time with you. Our parties, Friday drinks and chats are unforgettable moments. I am happy I met such great people! Moi Rodzice, Aga i Paweł, dziękuję Wam za dodawanie mi otuchy i wiarę we mnie. Dziękuję za to, że zawsze ciepło mnie witaliście kiedy wracałam do rodzinnego domu. Mamo, tato, wiem, że przyjazd do Belgii to dla Was duże przeżycie, tym bardziej dziękuję za to, że sie zdecydowaliście. Ik wil graag de familie van Stan bedanken, vooral Annick en Filip, ik heb veel steun van jullie gekregen. Jullie hebben me heel warm ontvangen. Ik dank jullie hartelijk voor jullie gastvrijheid, interesse en vriendschap. Stan, moje Kochanie, I want to thank you for your support, understanding and for being there for me. I am really happy that I met you that evening at Brahim’s. You always believed in me and tried to encourage me during hard times. Dziękuję for all the wonderful days we had, all the great moments we have now and I am looking forward to the future with you! Merci! Bedankt! Dziękuję! Thank you! vi List of abbreviations ACN Acetonitrile ATCC American Type Culture Collection BCCM Belgian Coordinated Collections of Micro-organisms BOX-PCR BOX-A1R-based repetitive extragenic palindromic-PCR BLAST Basic Local Alignment Search Tool CDS Coding DNA Sequence CFBP Collection Française de Bactéries associées aux Plantes CHCA α-cyano-4-hydroxycinnamic acid Cmi Clavibacter michiganensis subsp. insidiosus Cmm Clavibacter michiganensis subsp. michiganensis Cmn Clavibacter michiganensis subsp. nebraskensis Cms Clavibacter michiganensis subsp. sepedonicus Cmt Clavibacter michiganensis subsp. tessellarius CMR Comprehensive Microbial Resource CRISPR Clustered Regularly Interspaced Short Palindromic Repeats CWDE Cell Wall Degrading Enzyme dpi days post inoculation DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen EDGAR Efficient Database framework for comparative Genome Analyses using BLAST score Ratios EDTA Ethylene Diamine Tetraacetic Acid ELISA Enzyme-Linked Immunosorbent Assay EPPO European and Mediterranean Plant Protection Organization EPS Extracellular Polysaccharide ERIC-PCR Enterobacterial Repetitive Intergenic Consensus-PCR EU European Union FAME Fatty Acid Methyl Esters FAO Food and Agriculture Organization of the United Nations HGDI Hunter-Gaston Diversity Index HGT Horizontal Gene Transfer HR Hypersensitive Response ILVO Instituut voor Landbouw- en VisserijOnderzoek IS Insertion Sequence IS-LM-PCR Insertion Sequence Ligation-Mediated PCR ISF International Seed Federation ISSR Inter-Simple Sequence Repeat ITS Internal Transcribed Spacer region LMG Laboratory for Microbiology Ghent MALDI-TOF MS Matrix-Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry MCL Maximum Composite Likelihood model MEGA Molecular Evolutionary Genetics Analysis software ML Maximum Likelihood MLST Multi Locus Sequence Typing MLVA Multilocus Variable-Number Tandem Repeats Analysis vii MP Mate-Paired MP Maximum Parsimony MST Minimum Spanning Tree MTNA Mannitol, Trimethoprim, Nalidixic acid, Amphotericin medium NCBI National Centre for Biotechnology Information NCPPB National Collection of Plant Pathogenic Bacteria NJ Neighbor-Joining OEPP Organisation Européenne et Méditerranéenne pour la Protection des Plantes PAI Pathogenicity Island PAMPs Pathogen-Associated Molecular Patterns PCR Polymerase Chain Reaction NVWA Bacterial collection (Wageningen, The Netherlands) PDDCC Plant Diseases Division Culture Collection PE