Microbial assemblage in grapevine’s phyllosphere : who is the driver ? Prashant Singh To cite this version: Prashant Singh. Microbial assemblage in grapevine’s phyllosphere : who is the driver ?. Agricultural sciences. Montpellier SupAgro, 2018. English. NNT : 2018NSAM0042. tel-02159427 HAL Id: tel-02159427 https://tel.archives-ouvertes.fr/tel-02159427 Submitted on 18 Jun 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE Pour obtenir le grade de Docteur Délivrée par le Centre International d’Études Supérieures en Sciences Agronomiques Montpellier L’école Doctorate Systèmes Intégrès en Biologie, Agronomie, Géosciences L’unité de recherche AGAP Spécialité : Biologie intégrative de la plante Présentée par Prashant SINGH Microbial Assemblage in Grapevine's Phyllosphere: Who is the Driver? Soutenue le 30 novembre 2018 devant le jury composé de Isabelle MASNEUF, Professeur, Rapporteur Bordeaux Sciences Agro Aziz AZIZ, Maître de conférences Rapporteur Université de Reims Robin DUPONNIOS, DR, IRD Examinateur Montpellier Sabine GALINDO, Professeur Examinateur Université de Montpellier Patrice REY, Professeur Président du Bordeaux Sciences Agro jury Patrice THIS, DR, INRA Director Montpellier Jean-Pierre PÉROS, DR, INRA Co-Director Montpellier Microbial Assemblage in Grapevine's Phyllosphere: Who is the Driver? Prashant Singh M.Tech, Indian Institute of Technology, Kharagpur A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UMR1334 AGAP AMÉLIORATION GÉNÉTIQUE ET ADAPTATION DES PLANTES MÉDITERRANÉENNES ET TROPICALES INRA-SUPAGRO 2018 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. Je déclare par la présente que cette thèse est mon travail original et qu'il a été écrit par moi en son intégralité. J'ai dûment reconnu toutes les sources d'information qui ont été utilisé dans la thèse. Cette thèse n'a pas non plus été soumise dans aucune université auparavant. _______________________________________________________________ Prashant Singh 12 December, 2018 Dedicated to Phyllosphere, a generous niche supporting the microbial life, for their contributions to plants and mankind Acknowledgements The duration spent in France for my Ph.D. will always be one of the most memorable phases of my life. This phase has helped me to grow not only as a researcher but also as a person. I, therefore, take this opportunity to thank important people who made the wonderful journey possible. First and foremost, I would like to thank my supervisors Dr. Patrice This and Jean-Pierre Péros for choosing me for this challenging project and their immense guidance and support. Their inputs during the course of my thesis have made me a better researcher. I would also like to thank Sylvain Santoni, Audrey Weber, and Valerie Laucou for their constant support in lab work, experimental designs and Philippe Chatelet for his concrete suggestions for manuscript preparations. I am also thankful to European Union for funding this project via MicroWine network. I made new and quality friends via this collaborative network whose support had been a crucial part of my learning experience during my Ph.D. I would like to convey my special thanks to my lovely wife Kalpana. This journey would not have been possible without her unconditional love and support. Finally, I would like to especially thank my Parents, family members, friends from NIT Raipur and IIT Kharagpur and the owners of my rented apartment in Montpellier Peggy and Lattif, whose constant love and support has helped the 3 years of my stay at Montpellier joyful. Contents Contents ..................................................................................................................................... i Thesis summary ...................................................................................................................... iv Résumé de la thèse .......................................................................................................... v Chapter 1 .................................................................................................................................. 1 1.1. Introduction ...................................................................................... 2 1.1. Grapevines ....................................................................................... 2 1.2. Plant associated Microbiome........................................................... 3 1.2.1 Rhizosphere.................................................................................... 4 1.2.2. Endosphere ................................................................................... 4 1.2.3. Phyllosphere ................................................................................. 4 1.3. Characterisation of plant-associated microbiome ........................... 8 1.3.1. Culturable vs Non-culturable …................................................... 6 1.3.2. Next Generation Sequencing (NGS)............................................. 9 1.3.3. Target genes................................................................................... 11 1.3.4. Bioinformatic data analysis …...................................................... 13 1.3.4.1. Metagenomic Rapid Annotations using Subsystems Technology (MG-RAST) …............................................................................................................ 13 1.3.4.2. Quantitative Insights into Microbial Ecology (QIIME): …...... 13 1.3.4.3. MOTHUR ................................................................................. 14 1.3.4.4. UPARSE: highly accurate OTU sequences from microbial amplicon reads …............................................................................................................. 14 1.3.4.5. Divisive Amplicon Denoising Algorithm (DADA) …............... 15 1.4. Scope of the thesis ........................................................................... 17 1.4.1. Objectives …................................................................................. 17 1.4.2. Organization …............................................................................. 17 1.5. References …....................................................................................18 Chapter 2 Assessing the impact of plant genetic diversity in shaping the microbial community structure of Vitis vinifera phyllosphere in the Mediterranean (Published Manuscript) ....................................................................................... 25 2.1 Abstract ............................................................................................ 27 2.2 Introduction ..................................................................................... 27 2.3 Material and Methods ..................................................................... 28 2.4 Results ............................................................................................. 30 i 2.5 Discussion …................................................................................... 33 2.6 Acknowledgements …..................................................................... 36 2.7 Disclosure Statement …................................................................... 36 2.8 Funding …..................................................................................... 36 2.9 References ..................................................................................... 37 Chapter 3 Genotype-Environment Interaction Shapes the Microbial Assemblage in Grapevine’s Phyllosphere and Carposphere: An NGS Approach (Published Manuscript) ....................................................................................... 39 3.1 Abstract .......................................................................................... 40 3.2 Introduction .................................................................................... 40 3.3 Material and Methods .................................................................... 41 3.4 Results ............................................................................................ 43 3.5 Discussion ….................................................................................. 47 3.6 Conclusion …................................................................................. 48 3.7 Author Contribution ….................................................................. 48 3.8 Acknowledgements …................................................................... 48 3.9 Funding ......................................................................................... 48 3.10 References …............................................................................... 48 3.11 Supplementary Informations …................................................... 51 Chapter 4 Understanding the phyllosphere microbiome assemblage in grape-species with