Étude 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

Histological and molecular approaches for resistance to Fusarium oxysporum f.sp. radicis-vanillae, causal agent of root and stem rot in Vanilla spp. (Orchidaceae)

• Auteur / Autrice : Sayuj Koyyappurath
• Direction : Pascale Besse
• Type : Thèse de doctorat
• Discipline(s) : Biologie végétale
• Date : Soutenance le 08/07/2015
• Etablissement(s) : La Réunion
• Ecole(s) doctorale(s) : École doctorale Sciences, Technologies et Santé (Saint-Denis, La Réunion)
• Partenaire(s) de recherche : Laboratoire : Peuplements végétaux et bioagresseurs en milieu tropical (Saint-Pierre, Réunion)
• Jury : Président / Présidente : Thierry Pailler
• Jury : Examinateurs / Examinatrices : Christian Steinberg, Michel Grisoni
• Jury : Rapporteurs / Rapporteuses : Quirico Migheli, Altus Viljoen

Mots clés

Mots clés contrôlés

Relations plante-microorganisme pathogène

Transcriptome

Phylogénie moléculaire

Vanilliers

Fusarioses

Fusarioses

Lignine

Mots clés libres

Ile de La Réunion

Analyse Phylogénique

Lignine

Microscopique multiphoton

Pouvoir pathogène

Analyse spectrale

Transcriptome

Reunion Island

Phylogenetic analysis

Lignin

Multiphoton microscopy

Pathogenic power

Résumé

La production de vanille (Vanilla planifolia, Orchidaceae) est limitée par la pourriture des racines et des tiges (PRT) provoquée par des champignons telluriques du genre Fusarium contre lesquels la lutte chimique ou prophylactique est inefficace. Ce travail décrit finement l'interaction entre les vanilliers et les Fusarium dans le but de développer la lutte génétique. L'analyse moléculaire (gène EF1α et IGS) et la détermination du pouvoir pathogène de 365 souches de Fusarium isolées de vanilliers à La Réunion et à Madagascar ont démontré que les souches pathogènes sur vanillier appartiennent presque toutes au complexe d'espèce F. oxysporum et ont une origine polyphylétique. Les observations en microscopie à champ large et multi-photons démontrent que la colonisation des racines débute au niveau des poils absorbants puis gagne le cortex, mais que le système vasculaire est épargné. Cela conduit à renommer l'agent pathogène en F. oxysporum f. sp. radicis-vanillae (Forv). Dans les interactions incompatibles (souche pathogène et vanillier résistant), l'épaississement et la lignification des parois de l'hypoderme, constitutive et induite par le champignon, joue un rôle significatif dans le blocage de l'invasion mycélienne. Le rôle important des composés phénoliques dans la résistance des vanilliers a été confirmé par les analyses d'expression différentielle de transcrits obtenus de novo par RNA-seq Illumina. Enfin, un test robuste de détermination in-vitro de la résistance à Forv a été validé et de nouvelles sources de résistance génétique à la PRT ont été identifiées. Nos résultats ouvrent des perspectives prometteuses pour la sélection de variétés améliorées de vanilliers.

Vanilla is a high value cash crop that is continuously demanded by the agri-food and cosmetics industries for its incomparable flavor. Most of vanilla comes from the cured fruits of V. planifolia G. Jackson, a hemi-epiphytic climbing orchid cultivated in the humid tropics. In all the countries were it is cultivated, the vanilla vines suffer from a root and stem rot (RSR) caused by the soil borne fungus Fusarium oxysporum which dramatically reduces plant production and the durability of plantations. No efficient control method is currently available for this disease. Sources of genetic resistance to RSR exist in few vanilla relatives, but so far no commercial resistant variety has been produced. The purpose of this thesis was to better describe the diversity and histopathology of the causal agent of RSR and to evaluate the potential sources of genetic resistance that could be used in breeding programs. In a first step, a collection of 377 single-spored Fusarium isolates recovered from rotten roots and stems during surveys conducted in 52 vanilla plots from Reunion Island, Madagascar and French Polynesia were characterised. Representative subsets of isolates were genotyped using the Elongation Factor 1α and Intergenic Spacer gene sequences. Their pathogenicity was assayed by root dip inoculation on the susceptible V. planifolia accession pla0001. Results showed that F. oxysporum was the principal species responsible for the disease in the field, although a few F. solani isolates showing slight pathogenicity were also isolated. Fusarium oxysporum isolates were highly polyphyletic regardless of geographic origin or pathogenicity. Remarkably, their pathogenicity varied in gradient between non- pathogenic (about 42% of isolates) to highly pathogenic (14%). In a second step, 254 vanilla accessions comprising 18 species and six types of hybrids were assessed for resistance to RSR in the field (natural inoculum) and in the lab (in-vitro plants inoculated with Fo072). The strong resistance to RSR of all V. pompona accessions and hybrids of V. planifolia X V. pompona or V. phaeantha, were confirmed, and novel sources of resistance to RSR were added including, V. bahiana, V. costariciensis and V. crenulata. Most of the V. planifolia accessions, V. ×tahitensis and V. odorata were susceptible to RSR. However, three inbreeds of V. planifolia showed a high level of resistance to Forv. To our knowledge this is the first report of resistance to RSR in V. planifolia accessions. For the 26 accessions evaluated in both conditions, a strong correlation was observed between long term (9 years) evaluation in the field and ratings on in-vitro plants at 15dpi. Thirdly, we monitored by wide field and multiphoton microscopy the root infection process and the responses of one susceptible accession (V. planifolia pla0001) and two resistant accessions (V. planifolia pla0020 and V. pompona pom0018) to challenge inoculation with the severe isolate Fo072. In the compatible interaction (Fo072 – pla0001) invasion started from penetration of hyphae emitted from germinated conidia in the hairy region of root rapidly colonizing the cortex but never expanded to the vascular bundles up to the 9th dpi. It was therefore suggested to prefix the forma specialis name of the causal agent of RSR with radicis to point out its non-vascular pathogenicity in vanilla. In the two incompatible interactions, the important role played by hypodermis cells for impeding the invasion of the cortex by Fo072 was demonstrated by specific staining and spectral analysis of lignin precursors. Both constitutive and pathogen induced defense mechanism were described in pla0020 and pom0018. The mechanisms included the deposition of lignin in the hypodermal cell wall, entrapment of hyphae in specific hypodemal cells and polyphenolics secretion in intercellular spaces. Further, a de novo transcriptome analysis was experimentedon 8 pooled samples.