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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

By Sayuj Koyyappurath

Abstract

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 Inter genic 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%). I n 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 experimented on 8 pooled samples. Preliminary transcriptomic data were obtained by de novo RNAseq (Illumina) for eight combinations of resistant vs susceptible accessions, inoculated or not with pathogenic or non-pathogenic isolates. Analysis of the transcripts yielded 169 genes differentially expressed genes including of few genes involved in the plant resistance mechanisms and in the production of secondary metabolites and lignin biosynthesis. Altogether, our data clarified the aetiology of RSR, broadened the sources of resistance to Forv and enlightened the resistance mechanism at work in resistant genotypes. As such, they will enhance the breeding programs aimed at developing novel vanilla varieties resistant to RSR. (Résumé d'auteur

Topics: F30 - Génétique et amélioration des plantes, H20 - Maladies des plantes
Publisher: Université de la Réunion
Year: 2015
OAI identifier: oai:agritrop.cirad.fr:580789
Provided by: Agritrop

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