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In Vanilla Spp UNIVERSITE DE LA REUNION Faculté des Sciences et Technologies UMR « Peuplements Végétaux et Bio-agresseurs en Milieu Tropical » CIRAD – Université de la Réunion THESE Pour obtenir le diplôme de DOCTORAT Discipline : Sciences Formation Doctorale : Biologie Végétale Ecole Doctorale Sciences Technologies Santé Etude histo-pathologique et moléculaire de la résistance des vanilliers (Vanilla spp., Orchidaceae) à Fusarium oxysporum f.sp. radicis-vanillae, agent de la pourriture des racines et des tiges par Sayuj KOYYAPPURATH Soutenance publique du 08 Juillet 2015, devant un jury composé de : Professeur, Université de la Thierry PAILLER Président Réunion Professeur, Université de Quirico MIGHELI Rapporteur Sassari (Italie) Professeur, Université de Altus VILJOEN Rapporteur Stellenbosch (Afrique du Sud) Christian STEINBERG Directeur de Recherche, INRA Examinateur Michel GRISONI Chercheur, CIRAD Encadrant Professeur, Université de la Pascale BESSE Directrice de thèse Réunion Cette thése a reçu le soutien financier de la Région Réunion UNIVERSITE DE LA REUNION Faculté des Sciences et Technologies UMR « Peuplements Végétaux et Bio-agresseurs en Milieu Tropical » CIRAD – Université de la Réunion THESE Pour obtenir le diplôme de DOCTORAT Discipline : Sciences Formation Doctorale : Biologie Végétale Ecole Doctorale Sciences Technologies Santé Histological and molecular approaches for resistance to Fusarium oxysporum f.sp. radicis-vanillae, causal agent of root and stem rot in Vanilla spp. (Orchidaceae) by Sayuj KOYYAPPURATH Soutenance publique du 08 Juillet 2015, devant un jury composé de : Professor, University of La Thierry PAILLER President Reunion Professor, University of Quirico MIGHELI Rapporteur Sassari, Italy Professor, Stellenbosch Altus VILJOEN Rapporteur University, South Africa Christian STEINBERG Research Director, INRA Examinator Michel GRISONI Scientist, CIRAD Tutor Professor, University of La Pascale BESSE Thesis Director Reunion This thesis has received financial support from the Region Réunion Table of contents ACKNOWLEDGEMENTS ........................................................................................... 1 ABSTRACT ................................................................................................................... 1 CHAPTER I INTRODUCTION .................................................................................. 4 I Vanilla .................................................................................................................. 5 I-1 History of vanilla cultivation ........................................................................ 5 I-2 Economic importance of vanilla ................................................................... 7 I-3 Taxonomy of Vanilla .................................................................................... 8 I-4 Botany of Vanilla .......................................................................................... 9 I-5 Histology of vanilla ..................................................................................... 11 I-6 Agronomy of Vanilla .................................................................................. 13 I-7 Diseases of vanilla ...................................................................................... 15 I-8 Root and stem rot of Vanilla ....................................................................... 17 II Fusarium oxysporum .......................................................................................... 18 II-1 Taxonomy of Fusarium oxysporum ............................................................ 19 II-2 Morphological characteristics ..................................................................... 19 II-3 Diversity of Fusarium ................................................................................. 20 II-4 Pathogenicity of Fusarium oxysporum ....................................................... 22 II-5 Infection pattern .......................................................................................... 24 II-6 Fungal genes responsible for pathogenicity ................................................ 24 III Plant pathogen interactions ............................................................................. 28 III-1 Plant recognition ........................................................................................ 28 III-2 R gene mediated resistance ........................................................................ 29 IV Control of RSR of vanilla ............................................................................... 32 IV-1 Agronomical management ......................................................................... 33 IV-2 Chemical control ........................................................................................ 33 IV-3 Biocontrol agents ....................................................................................... 34 IV-4 Genetic resistance ...................................................................................... 34 V Aim and Objectives of the study .................................................................... 35 CHAPTER II Etiology and diversity of RSR and diversity of the causal agent ....... 38 I Introduction ........................................................................................................ 41 II Materials and Methods ....................................................................................... 43 II-1 Sample collection ........................................................................................ 43 II-2 Isolation, purification and morphological identification of Fusarium isolates 43 II-3 DNA extraction ........................................................................................... 44 II-4 PCR amplification and DNA sequencing ................................................... 44 II-5 Pathogenicity tests ...................................................................................... 45 II-6 Histological observations ............................................................................ 46 II-7 Data analysis ............................................................................................... 46 III Results ............................................................................................................ 47 III-1 Field survey and symptomatology ............................................................. 47 III-2 Recovery of fungal isolates ........................................................................ 48 III-3 Molecular diversity analysis ...................................................................... 49 III-4 Morphological identification ...................................................................... 50 III-5 Pathogenicity assessment ........................................................................... 51 III-6 Histopathological observations .................................................................. 53 IV Discussion ....................................................................................................... 53 V Acknowledgements ........................................................................................ 58 VI References ...................................................................................................... 58 VII Figures captions and Tables ........................................................................... 62 CHAPTER III Differential responses of vanilla accessions to root and stem rot ...... 72 I Introduction ........................................................................................................ 76 II Materials and Methods ....................................................................................... 78 II-1 Fungal isolates and vanilla genotypes ......................................................... 78 II-2 Evaluation of RSR resistance in field conditions ....................................... 79 II-3 In-vitro plant inoculation and assay ............................................................ 79 II-4 Wide field microscopy ................................................................................ 79 II-5 Multi-photon confocal microscopic analysis .............................................. 80 II-6 Emissions spectral analysis ......................................................................... 81 II-7 Data analysis ............................................................................................... 82 III Results ............................................................................................................ 82 III-1 Field evaluation of accessions .................................................................... 82 III-2 In-vitro germplasm screening for resistance to Forv ................................. 83 III-3 Correlations between field and in-vitro evaluations of RSR resistance ..... 84 III-4 Colonization of the root surface ................................................................. 85 III-5 Colonization in the root tissues .................................................................. 85 III-6 Anatomical differences between susceptible and resistant cultivars ......... 86 IV Discussion ....................................................................................................... 88 IV-1 Forv infection and colonization pattern ..................................................... 88 IV-2 Cellular and histochemical mechanisms in Forv resistance ...................... 89 IV-3 Screening for
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