diagnostics for quarantine plant pathogenic .diagnostics for quarantine plant pathogenic clavibacter

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  • 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 Zauga

    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

  • ii

    Joanna Zauga Diagnostics for quarantine plant pathogenic Clavibacter and relatives: molecular

    characterization, identification and epidemiological studies

    Copyright 2013 Joanna Zauga


    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


    Publicly defended in Ghent, Belgium, October 11th, 2013

  • iii

    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 Hfte 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

  • v


    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 Hfte, 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.

  • vi

    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! Dziki za wszystko moja najlepsza koleanko!

    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 Pawe, dzikuj Wam za dodawanie mi otuchy i wiar we mnie. Dzikuj za to, e

    zawsze ciepo mnie witalicie kiedy wracaam do rodzinnego domu. Mamo, tato, wiem, e przyjazd

    do Belgii to dla Was due przeycie, tym bardziej dzikuj za to, e sie zdecydowalicie.

    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 Brahims. You always believed in me and tried

    to encourage me during hard times. Dzikuj for all the wonderful days we had, all the great

    moments we have now and I am looking forward to the future with you!




    Thank you!

  • vii

    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 Franaise de Bactries associes aux Plantes CHCA -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 R


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