ME T H O D S I N MO L E C U L A R B I O L O G Y

Series EditorJohn M. Walker

School of Life and Medical SciencesUniversity of Hertfordshire

Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:http://www.springer.com/series/7651

Host-Pathogen Interactions

Methods and Protocols

Edited by

Carlos Medina

Departamento de Biología Molecular e Ingeniería Bioquímica, Centro Andaluz de Biología delDesarrollo, CSIC/Universidad Pablo de Olavide/Junta de Andalucía

Seville, SpainDepartamento de Microbiología, Facultad de Biología, Universidad de Sevilla

Seville, Spain

Francisco Javier López-Baena

Department of Microbiology, University of SevilleSeville, Spain

EditorsCarlos MedinaDepartamento de Biologıa Molecular eIngenierıa Bioquımica, CentroAndaluz de Biologıa del DesarrolloCSIC/Universidad Pablo deOlavide/Junta de AndalucıaSeville, Spain

Departamento de Microbiologıa, Facultad de BiologıaUniversidad de SevillaSeville, Spain

Francisco Javier Lopez-BaenaDepartment of MicrobiologyUniversity of SevilleSeville, Spain

ISSN 1064-3745 ISSN 1940-6029 (electronic)Methods in Molecular BiologyISBN 978-1-4939-7603-4 ISBN 978-1-4939-7604-1 (eBook)https://doi.org/10.1007/978-1-4939-7604-1

Library of Congress Control Number: 2017962284

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Foreword

Colonization of eukaryotic hosts by bacteria causes disease in certain cases, and the distinc-tion between commensalism, symbiosis, and opportunistic pathogenesis is blurred. Even anencounter with a primary pathogen has an unpredictable outcome and does not necessarilycause disease. This uncertainty is due to the complexity of bacterium-host interactions,which involve multiple factors, both bacterial and eukaryotic. Microbe-microbe interactionsintroduce further complexity into this scenario.

Genetic and molecular analysis has proven useful to dissect bacterium-host interactionsin animals and plants, and in the last few decades has been enriched with novel approachessuch as bioinformatics, single cell analysis, live imaging technology, and mathematicalmodeling. Aside from deepening our understanding of infectious diseases, multidisciplinaryknowledge may inspire novel therapeutic strategies. For instance, identification of virulencefunctions may identify targets for future antibacterial drugs. In turn, the limitations andfailures of antibacterial drugs may revive the one-century-old interest in phage therapy.

This book deals with bacterial-host interactions in pathogens and symbionts of humansand plants and describes state-of-the-art methods used in their study. Reviews of reduction-ist analysis in the laboratory are accompanied by descriptions of host-pathogen interactionsin vivo and at the systems biology level. Altogether, the chapters of this book provide abroad, multidisciplinary view of bacterial pathogenesis from the twenty-first century per-spective. The book also contains glimpses into the future of the field, with implications inhuman health and in agriculture.

Department of Genetics, Faculty of Biology Josep CasadesusUniversity of Seville, Seville, Spain

v

Preface

We live immersed in a microbial world where microorganisms compose around 30% of theestimated living biomass. It has been proposed that the first life forms appeared on ourplanet around 3900 million years ago, and over the course of Earth’s history, they have beenspreading and evolving. Recent estimations propose the existence of up to one trillion (1012)microbial species, entailing a greater biodiversity than microbiologists could ever imagine.Microbes have colonized almost every niche of the planet, from the deepest ocean trenchesto high layers of the atmosphere. Thus, they have adapted their metabolism to survive inhabitats as extreme as boiling acid, temperatures below the pure water congelation point, orin desiccant spaces such as salt crystallizer ponds.

Subsequently to the appearance of complex forms of life, microbes developed numerousmolecular adaptations to expand their ecological niches and live in close relation with them.These interactions could be favorable when the presence of the microbe contributes to theprofit of its interactor, or harmful when the higher organism is damaged as a result. In anycase, microbes living inside their hosts have learnt to avoid immune responses or drive themto their own benefit. According to this, the study of the interaction between superioreukaryotes (plants and animals) and microbes has become essential from environmental,economical, or health points of view. In the particular case of human pathogens, thedeciphering of how such microorganisms evade immune responses or even resist conven-tional antibiotic treatments brings in an immediate urgency since the mortality due to lethalinfections is predicted to become the leading cause of death by 2050. Therefore, theunderstanding of how host-pathogen interactions take place at the molecular, organism,or population level will help to discover the preventive measurements that should be taken inmind to avoid such diseases.

In a different context, the injurious interactions of microorganisms with plants forhuman or animal feeding deserve special attention since millions of tons per year are lostdue to diverse microbial plagues. The economic impact of this wastage, aggravated by thereduction in the permissions for chemicals that can be applied to crops to fight plantpathogens, highlights the importance of host-pathogen research since it can shed light onthe knowledge of these interactions to predict how an infection could occur. Besides thisconcern, a consideration of beneficial microorganisms must be kept in mind while itsapplication to crops can improve their yield by means of a wide variety of plant growth-promoting activities, ranging from increasing the availability of nutrients for plants tocounteracting the negative effect of specific pathogens with biocontrol activities.

In this book, we propose numerous multidisciplinary approaches employed to analyzethe role of different molecules or strategies used by different guests to survive inside hosts.

The manual is mainly divided into two sections, depending on the nature of the hostorganism, which can be an animal or a plant host, and includes molecular biology andbiochemical techniques necessary to study these host-pathogen interactions.

With respect to animal-microbial pathogen interactions, we have included several chap-ters focused on Salmonella, which is a bacterium responsible for one of the most commonfoodborne diseases in the world and is also used as a model of pathogenicity for interactionswith animal cells. Thus, different experimental approaches to dissect the regulation ofSalmonella virulence genes at transcriptional and translational levels are shown. Additionally,

vii

and taking advantage of interesting Salmonella characteristics, such as the capacity of thisbacterium to specifically colonize tumoral tissues, it has been projected that the controlleduse of this bacterium could be used as an antitumoral tool. In parallel, the use of three-dimensional living models, such as organoids, to study the behavior of another gastrointes-tinal pathogen, Helicobacter pylori, is described in Chapter 1.

Other authors have highlighted the increasing importance of the use of systems biology,bioinformatics, and other -omics technologies to create models to study genetic associationsin host-pathogen interactions or predict interactions between microbial proteins and effec-tors and their host targets.

Finally, three review chapters focused on animal-pathogen interactions are included. Inone of them, the use of genetic association studies for the identification of alleles involved inthe infection process is shown. In addition, the use of several molecular biology tools forpathogens genotyping, a valuable tool for the identification of bacterial strains as well as forepidemiological surveillance, is also discussed. Moreover, one interesting chapter providessome recommendations about the use of phage therapy as an alternative with high potentialto treat diseases caused by bacterial agents.

This book also presents some techniques to study microbe-microbe relationships, sincemicrobes have developed successful strategies, including bacteria-bacteria communicationthrough quorum sensing systems, to regulate traits associated with the coordinated infec-tion of higher organisms or the formation of biofilms to increase bacterial survival withinthe host.

Chapters included in the plant–pathogen interactions section describe tools to studyvirulence genes expression, techniques to identify and characterize effectors secretedthrough the type 3 secretion system, assays to determine phenotypes associated with plantpathogenicity, analysis of images using confocal microscopy, screening of surface polysac-charides, such as exopolysaccharides, regulated by second messengers, or the identificationof small RNAs involved in the regulation of many bacterial traits associated with virulence.

Since below-ground plant-pathogen interactions are of increasing interest in recentyears, a chapter is dedicated to a nondestructive split-root system designed to monitorroot infection that can also be used to study plant signals, triggered by microbes andgenerated in the roots, which can be delivered to other roots or to above-ground tissues.Finally, considering that plant pathogens must share the space with other microbial strains,plant growth-promoting activities of rhizosphere microorganisms are also included in thisbook. These activities have a potential impact not only on plant growth but also in thesurvival of plant pathogen populations.

In summary, this book provides a huge number of molecular and bioinformaticstechniques used in reputed laboratories of different countries all around the world, helpfulfor different kind of host-pathogen interactions studies.

Seville, Spain Francisco Javier L�opez-BaenaCarlos Medina

viii Preface

Contents

Foreword. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vPreface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viiContributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

1 Genetic Association Studies in Host–Pathogen Interaction Analysis . . . . . . . . . . . 1Jose Luis Royo and Luis Miguel Real

2 Bacterial Genotyping Methods: From the Basics to Modern. . . . . . . . . . . . . . . . . . 13Margarita M. Ochoa-Dıaz, Silvana Daza-Giovannetty,and Doris G�omez-Camargo

PART I ANIMAL-PATHOGEN INTERACTION SECTION

3 Real-Time Reverse Transcription PCR as a Tool to Study VirulenceGene Regulation in Bacterial Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Gili Aviv and Ohad Gal-Mor

4 Usage of a Bioluminescence Reporter System to Image PromoterActivity During Host Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Gili Aviv and Ohad Gal-Mor

5 lacZ Reporter System as a Tool to Study Virulence Gene Regulationin Bacterial Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39Gili Aviv and Ohad Gal-Mor

6 Western Blotting Against Tagged Virulence Determinants to StudyBacterial Pathogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Gili Aviv and Ohad Gal-Mor

7 Molecular Methods to Analyze the Effect of Proteins Expressedby Salmonella During Its Intracellular Stage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55Carlos Medina, Beatriz Mesa-Pereira, Eva M. Camacho, Amando Flores,and Eduardo Santero

8 Organoids as a Model to Study Infectious Disease . . . . . . . . . . . . . . . . . . . . . . . . . . 71Kristen A. Engevik, Andrea L. Matthis, Marshall H. Montrose,and Eitaro Aihara

9 Surface Proteome Biotinylation Combined with Bioinformatic Toolsas a Strategy for Predicting Pathogen Interacting Proteins . . . . . . . . . . . . . . . . . . . 83Anita Horvatic, Josipa Kules, Nicolas Guillemin, Franjo Martinkovic,Iva Stimac, Vladimir Mrljak, and Mangesh Bhide

10 Systems Biology Modeling to Study Pathogen–Host Interactions . . . . . . . . . . . . . 97Muberra Fatma Cesur and Saliha Durmus

11 Phage Therapy: Various Perspectives on How to Improve the Art . . . . . . . . . . . . 113Stephen T. Abedon

ix

PART II MICROBE-MICROBE INTERACTION SECTION

12 Application of RNA-seq and Bioimaging Methods to StudyMicrobe–Microbe Interactions and Their Effects on BiofilmFormation and Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131Cristina Isabel Amador, Claus Sternberg, and Lars Jelsbak

13 Serial Dilution-Based Growth Curves and Growth Curve Synchronizationfor High-Resolution Time Series of Bacterial Biofilm Growth . . . . . . . . . . . . . . . . 159Fernando Govantes

14 Detection of Bacterial Quorum Sensing Molecules. . . . . . . . . . . . . . . . . . . . . . . . . . 171Elke Stein and Adam Schikora

PART III PLANT-PATHOGEN INTERACTION SECTION

15 Generating Chromosome-Located Transcriptional Fusionsto Fluorescent Proteins for Single-Cell Gene Expression Analysisin Pseudomonas syringae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183Jose S. Rufian, Diego L�opez-Marquez, Nieves L�opez-Pagan,Murray Grant, Javier Ruiz-Albert, and Carmen R. Beuz�on

16 Introduction of Genetic Material in Ralstonia solanacearumThrough Natural Transformation and Conjugation . . . . . . . . . . . . . . . . . . . . . . . . . 201Anthony Perrier, Patrick Barberis, and Stephane Genin

17 In Vitro and In Vivo Secretion/Translocation Assays to IdentifyNovel Ralstonia solanacearum Type 3 Effectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209Fabien Lonjon, Nemo Peeters, Stephane Genin, and Fabienne Vailleau

18 Plant Pathogenicity Phenotyping of Ralstonia solanacearum Strains . . . . . . . . . . 223Arry Morel, Nemo Peeters, Fabienne Vailleau, Patrick Barberis,Gaofei Jiang, Richard Berthome, and Alice Guidot

19 Methods to Quantify Biotic-Induced Stress in Plants . . . . . . . . . . . . . . . . . . . . . . . . 241Marcel Bach-Pages and Gail M. Preston

20 From Sample to Data: Preparing, Obtaining, and Analyzing Imagesof Plant-Pathogen Interactions Using Confocal Microscopy. . . . . . . . . . . . . . . . . . 257Helen N. Fones and George R. Littlejohn

21 Screening of c-di-GMP-Regulated Exopolysaccharides in HostInteracting Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263Jochen Schmid, Broder Ruhmann, Volker Sieber, Lorena Romero-Jimenez,Juan Sanjuan, and Daniel Perez-Mendoza

22 Primary Characterization of Small RNAs in SymbioticNitrogen-Fixing Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277Marta Robledo, Natalia I. Garcıa-Tomsig, and Jose I. Jimenez-Zurdo

23 A New, Nondestructive, Split-Root System for Localand Systemic Plant Responses Studies with Soybean. . . . . . . . . . . . . . . . . . . . . . . . . 297Angeles Hidalgo, Jose E. Ruiz-Sainz, and Jose M. Vinardell

x Contents

24 Methods for the Characterization of Plant-Growth PromotingRhizobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307Md. Motaher Hossain and Farjana Sultana

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329

Contents xi

Contributors

STEPHEN T. ABEDON � Department of Microbiology, The Ohio State University, Mansfield,OH, USA

EITARO AIHARA � Department of Pharmacology and Systems Physiology, University ofCincinnati, Cincinnati, OH, USA

CRISTINA ISABEL AMADOR � Department of Biotechnology and Biomedicine, TechnicalUniversity of Denmark, Lyngby, Denmark

GILI AVIV � The Infectious Diseases Research Laboratory, Sheba Medical Center,Tel-Hashomer, Ramat Gan, Israel; Department of Clinical Microbiology and Immunology,Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

MARCEL BACH-PAGES � Department of Plant Sciences, University of Oxford, Oxford, UKPATRICK BARBERIS � LIPM, Universite de Toulouse, INRA, CNRS, INPT, Castanet-Tolosan,

FranceRICHARD BERTHOME � LIPM, Universite de Toulouse, INRA, CNRS, INPT, Castanet-

Tolosan, FranceCARMEN R. BEUZON � Dpto. Biologıa Celular, Genetica y Fisiologıa, Instituto de

Hortofruticultura Subtropical y Mediterranea, Universidad de Malaga-Consejo Superiorde Investigaciones Cientıficas (IHSM-UMA-CSIC), Malaga, Spain

MANGESH BHIDE � ERA Chair VetMedZg Project, Internal Diseases Clinic, Faculty ofVeterinary Medicine, University of Zagreb, Zagreb, Croatia; Laboratory of BiomedicalMicrobiology and Immunology, University of Veterinary Medicine and Pharmacy, Kosice,Slovakia; Institute of Neuroimmunology, Slovakia Academy of Sciences, Bratislava,Slovakia

EVA M. CAMACHO � Departamento de Biologıa Molecular e Ingenierıa Bioquımica, CentroAndaluz de Biologıa del Desarrollo, CSIC/ Universidad Pablo de Olavide/ Junta deAndalucıa, Seville, Spain

MUBERRA FATMA CESUR � Computational Systems Biology Group, Department ofBioengineering, Gebze Technical University, Kocaeli, Turkey

SILVANA DAZA-GIOVANNETTY � Faculty of Medicine, Tropical Medicine Doctorate, UNIMOL,University of Cartagena, Cartagena, Colombia

SALIHA DURMUS � Computational Systems Biology Group, Department of Bioengineering,Gebze Technical University, Kocaeli, Turkey

KRISTEN A. ENGEVIK � Department of Pharmacology and Systems Physiology, University ofCincinnati, Cincinnati, OH, USA

AMANDO FLORES � Departamento de Biologıa Molecular e Ingenierıa Bioquımica, CentroAndaluz de Biologıa del Desarrollo, CSIC/ Universidad Pablo de Olavide/ Junta deAndalucıa, Seville, Spain

HELEN N. FONES � Biosciences, University of Exeter, Exeter, UKOHAD GAL-MOR � The Infectious Diseases Research Laboratory, Sheba Medical Center, Tel-

Hashomer, Ramat Gan, Israel; Department of Clinical Microbiology and Immunology,Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

NATALIA I. GARCIA-TOMSIG � Grupo de Ecologıa Genetica de la Rizosfera, Estaci�onExperimental del Zaidın (CSIC), Granada, Spain

STEPHANE GENIN � LIPM, Universite de Toulouse, INRA, CNRS, Castanet-Tolosan, France

xiii

DORIS GOMEZ-CAMARGO � Faculty of Medicine, Tropical Medicine Doctorate, UNIMOL,University of Cartagena, Cartagena, Colombia

FERNANDO GOVANTES � Centro Andaluz de Biologıa del Desarrollo, Universidad Pablo deOlavide, Consejo Superior de Investigaciones Cientıficas and Junta de Andalucıa, Seville,Spain

MURRAY GRANT � School of Life Sciences, University of Warwick, Conventry, UKALICE GUIDOT � LIPM, Universite de Toulouse, INRA, CNRS, INPT, Castanet-Tolosan,

FranceNICOLAS GUILLEMIN � ERA Chair VetMedZg Project, Internal Diseases Clinic, Faculty of

Veterinary Medicine, University of Zagreb, Zagreb, CroatiaANGELES HIDALGO � Department of Microbiology, Faculty of Biology, University of Seville,

Seville, SpainANITA HORVATIC � ERA Chair VetMedZg Project, Internal Diseases Clinic, Faculty of

Veterinary Medicine, University of Zagreb, Zagreb, CroatiaMD. MOTAHER HOSSAIN � Department of Plant Pathology, Bangabandhu Sheikh Mujibur

Rahman Agricultural University, Gazipur, BangladeshLARS JELSBAK � Department of Biotechnology and Biomedicine, Technical University of

Denmark, Lyngby, DenmarkGAOFEI JIANG � LIPM, Universite de Toulouse, INRA, CNRS, INPT, Castanet-Tolosan,

FranceJOSE I. JIMENEZ-ZURDO � Grupo de Ecologıa Genetica de la Rizosfera, Estaci�on Experimental

del Zaidın (CSIC), Granada, SpainJOSIPA KULES � ERAChair VetMedZg Project, Internal Diseases Clinic, Faculty of Veterinary

Medicine, University of Zagreb, Zagreb, CroatiaGEORGE R. LITTLEJOHN � School of Biological Sciences, Plymouth University, Plymouth, UKFABIEN LONJON � LIPM, Universite de Toulouse, INRA, CNRS, INPT, Castanet-Tolosan,

FranceDIEGO LOPEZ-MARQUEZ � Dpto. Biologıa Celular, Genetica y Fisiologıa, Instituto de

Hortofruticultura Subtropical y Mediterranea, Universidad de Malaga-Consejo Superiorde Investigaciones Cientıficas (IHSM-UMA-CSIC), Malaga, Spain

NIEVES LOPEZ-PAGAN � Dpto. Biologıa Celular, Genetica y Fisiologıa, Instituto deHortofruticultura Subtropical y Mediterranea, Universidad de Malaga-Consejo Superiorde Investigaciones Cientıficas (IHSM-UMA-CSIC), Malaga, Spain

FRANJO MARTINKOVIC � Department for Parasitology and Parasitic Diseases with Clinics,Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia

ANDREA L. MATTHIS � Department of Pharmacology and Systems Physiology, University ofCincinnati, Cincinnati, OH, USA

CARLOS MEDINA � Departamento de Biologıa Molecular e Ingenierıa Bioquımica, CentroAndaluz de Biologıa del Desarrollo, CSIC/ Universidad Pablo de Olavide/ Junta deAndalucıa, Seville, Spain; Departamento de Microbiologıa, Facultad de Biologıa,Universidad de Sevilla, Seville, Spain

BEATRIZ MESA-PEREIRA � Teagasc Food Research Centre, Teagasc Moorepark, Co. Cork,Ireland; APC Microbiome Institute, University College Cork, Cork, Ireland

MARSHALL H. MONTROSE � Department of Pharmacology and Systems Physiology, Universityof Cincinnati, Cincinnati, OH, USA

xiv Contributors

ARRY MOREL � LIPM, Universite de Toulouse, INRA, CNRS, INPT, Castanet-Tolosan,France

VLADIMIR MRLJAK � ERA Chair VetMedZg Project, Internal Diseases Clinic, Faculty ofVeterinary Medicine, University of Zagreb, Zagreb, Croatia

MARGARITAM. OCHOA-DIAZ � Faculty of Medicine, Tropical Medicine Doctorate, UNIMOL,University of Cartagena, Cartagena, Colombia

NEMO PEETERS � LIPM, Universite de Toulouse, INRA, CNRS, INPT, Castanet-Tolosan,France

DANIEL PEREZ-MENDOZA � Dpto. Microbiologıa del Suelo y Sistemas Simbi�oticos, Estaci�onExperimental del Zaidın, CSIC, Granada, Spain

ANTHONY PERRIER � LIPM, Universite de Toulouse, INRA, CNRS, Castanet-Tolosan, FranceGAIL M. PRESTON � Department of Plant Sciences, University of Oxford, Oxford, UKLUIS MIGUEL REAL � Unidad Clınica de Enfermedades Infecciosas y Microbiologıa, Hospital

Universitario de Valme, Seville, SpainMARTA ROBLEDO � Grupo de Ecologıa Genetica de la Rizosfera, Estaci�on Experimental del

Zaidın (CSIC), Granada, SpainLORENA ROMERO-JIMENEZ � Dpto. Microbiologıa del Suelo y Sistemas Simbi�oticos, Estaci�on

Experimental del Zaidın, CSIC, Granada, SpainJOSE LUIS ROYO � Departamento de Bioquımica, Biologıa Molecular e Inmunologıa,

Facultad de Medicina, Universidad de Malaga, Malaga, SpainJOSE S. RUFIAN � Dpto. Biologıa Celular, Genetica y Fisiologıa, Instituto de

Hortofruticultura Subtropical y Mediterranea, Universidad de Malaga-Consejo Superiorde Investigaciones Cientıficas (IHSM-UMA-CSIC), Malaga, Spain

BRODER RUHMANN � Chair of Chemistry of Biogenic Resources, Technical University ofMunich, Straubing, Germany

JAVIER RUIZ-ALBERT � Dpto. Biologıa Celular, Genetica y Fisiologıa, Instituto deHortofruticultura Subtropical y Mediterranea, Universidad de Malaga-Consejo Superiorde Investigaciones Cientıficas (IHSM-UMA-CSIC), Malaga, Spain

JOSE E. RUIZ-SAINZ � Department of Microbiology, Faculty of Biology, University of Seville,Seville, Spain

JUAN SANJUAN � Dpto. Microbiologıa del Suelo y Sistemas Simbi�oticos, Estaci�on Experimentaldel Zaidın, CSIC, Granada, Spain

EDUARDO SANTERO � Departamento de Biologıa Molecular e Ingenierıa Bioquımica, CentroAndaluz de Biologıa del Desarrollo, CSIC/ Universidad Pablo de Olavide/ Junta deAndalucıa, Seville, Spain

ADAM SCHIKORA � Julius Kuhn-Institut, Federal Research Centre for Cultivated Plants(JKI), Institute for Epidemiology and Pathogen Diagnostics, Braunschweig, Germany

JOCHEN SCHMID � Chair of Chemistry of Biogenic Resources, Technical University of Munich,Straubing, Germany

VOLKER SIEBER � Chair of Chemistry of Biogenic Resources, Technical University of Munich,Straubing, Germany

ELKE STEIN � Institute for Phytopathology, IFZ, Justus Liebig University Giessen, Gießen,Germany

CLAUS STERNBERG � Department of Biotechnology and Biomedicine, Technical Universityof Denmark, Lyngby, Denmark

IVA STIMAC � Department for Parasitology and Parasitic Diseases with Clinics, Facultyof Veterinary Medicine, University of Zagreb, Zagreb, Croatia

Contributors xv

FARJANA SULTANA � College of Agricultural Sciences, International University of BusinessAgricultural University, Dhaka, Bangladesh

FABIENNE VAILLEAU � LIPM, Universite de Toulouse, INRA, CNRS, INPT, Castanet-Tolosan, France

JOSE M. VINARDELL � Department of Microbiology, Faculty of Biology, University of Seville,Seville, Spain

xvi Contributors