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Intérêt De La Symbiose Mycorhizienne À Arbuscules Dans La Phytoremédiation Des Sols Historiquement Contaminés Par Les Hydro

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Intérêt De La Symbiose Mycorhizienne À Arbuscules Dans La Phytoremédiation Des Sols Historiquement Contaminés Par Les Hydro Intérêt de la symbiose mycorhizienne à arbuscules dans la phytoremédiation des sols historiquement contaminés par les hydrocarbures : de la protection à la dissipation Ingrid Lenoir To cite this version: Ingrid Lenoir. Intérêt de la symbiose mycorhizienne à arbuscules dans la phytoremédiation des sols historiquement contaminés par les hydrocarbures : de la protection à la dissipation. Biotechnologies. Université du Littoral Côte d’Opale, 2015. Français. NNT : 2015DUNK0398. tel-01552180v1 HAL Id: tel-01552180 https://tel.archives-ouvertes.fr/tel-01552180v1 Submitted on 1 Jul 2017 (v1), last revised 21 Sep 2017 (v2) 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. THESE DE L’UNIVERSITE DU LITTORAL COTE D’OPALE Pour obtenir le grade de DOCTEUR DE L’UNIVERSITE Ecole Doctorale des Sciences de la Matière, du Rayonnement et de l’Environnement Ingénierie des Fonctions Biologiques Mention: Physiologie végétale par Ingrid LENOIR Intérêt de la symbiose mycorhizienne à arbuscules dans la phytoremédiation des sols historiquement contaminés par les hydrocarbures: de la protection à la dissipation Soutenue le 9 juin 2015 devant la commission d’examen : CHALOT M., Professeur, Université de Franche-Comté, Montbéliard, France Rapporteur HIJRI M., Professeur, Université de Montréal, Montréal, Canada Rapporteur CRANENBROUCK S., Chargé de recherche, UCL, Belgique Examinateur DERAM A. Professeur, Université de Lille, Lille, France Examinateur LOUNES-HADJ SAHRAOUI A., Maître de conférences-HDR, ULCO, France Directrice de thèse FONTAINE J., Maître de conférences-HDR, ULCO, France Co-directeur de thèse Unité de Chimie Environnementale et Interactions sur le Vivant (UCEIV) 50, rue Ferdinand Buisson, BP 669 - 62228 Calais cedex Remerciements Remerciements Je commence par une pensée pour Monsieur Pirouz Shirali, Professeur à l’ULCO, éternel Directeur de l’UCEIV, qui était à l’écoute des étudiants. Je remercie également Monsieur Dominique Courcot, Professeur à l’ULCO, Directeur actuel de l’UCEIV, pour vos encouragements et votre oreille attentive. Je tiens à remercier vivement Madame Anissa Lounès-Hadj Sahraoui, Maître de conférences-HDR à l’ULCO et directrice de cette thèse. Je lui adresse ma reconnaissance pour m’avoir transmis son savoir et m’avoir guidé tout au long de cette thèse, tout en me laissant prendre des initiatives. Merci tout simplement de m’avoir fait confiance et de m’avoir poussé aux bouts de mes limites lorsque les difficultés me paraissaient insurmontables. Mes remerciements vont également à Jöel Fontaine, Maître de conférences-HDR à l’ULCO et co-directeur de thèse. Vous m’avez initié au monde de la recherche et de la mycorhize arbusculaire lors de mes stages de Master 1 et 2 à l’UCEIV. Vous m’avez également encadré et donné de précieux conseils tout au long de la thèse. Je remercie également Monsieur Michel Chalot, Professeur à l’Université de Franche-Comté et Monsieur Mohamed Hijri, Professeur à l’Université de Montréal, qui me font l’honneur d’évaluer mon travail et d’en être les rapporteurs. Ma gratitude va également à Madame Sylvie Cranenbrouck, Chargé de Recherche à l’Université Catholique de Louvain et Madame Annabelle Deram, Professeur à l’Université de Lille, pour avoir accepté de participer à ce jury. Je voudrais également remercier la Région Nord-Pas-de-Calais et le Pôle Métropolitain de la Côte d’Opale (PMCO) pour m’avoir octroyé la bourse de thèse. Je remercie Yolande Dalpé, Professeur associé à Agriculture et Agroalimentaire Canada pour l’identification des spores et notre discussion enrichissante sur les champignons mycorhiziens à arbuscules. Remerciements Un grand merci à Benoît Tisserand, Ingénieur de Recherche à l’UCEIV-Calais, pour l’initiation aux techniques de la biologie moléculaire, le nombre faramineux d’essais réalisés et de boîtes de Pétri repiquées ! Merci à Frédéric Laruelle, Ingénieur de Recherche à l’UCEIV-Calais, le pro de la chromatographie, pour l’identification des hydrocarbures et ses conseils techniques au quotidien. Merci à Anthony Verdin, Ingénieur d’Etudes à l’UCEIV- Dunkerque, pour sa disponibilité lorsque j’ai eu besoin du lecteur de plaques et ses bons tuyaux pour avoir des produits au rabais. Merci à ma chouchoute, Natacha Bourdon, Technicienne à l’UCEIV-Calais, qui m’a aidé au quotidien et surtout permis de travailler dans la joie et la bonne humeur (rires et chansons). Je remercie également l’ensemble des doctorants et post-doctorants, encore présents ou non : Sonia Labidi pour m’avoir appris à extraire les lipides et pour sa gentillesse, Ghalia et Hacène qui ont toujours été bienveillants à mon égard, ainsi que Maryline, Maréva, Christine, Sihem, Karima et Imad. Merci également à Giang qui m’a appris certaines choses en biologie moléculaire. Je souhaite remercier les membres de l’UCEIV-Calais pour les bons moments passés en votre compagnie. Je nomme Béatrice, Philippe, Maryline et surtout Corinne et Sandrine qui prennent les étudiants sous leurs ailes. Je remercie également mes amies et ma famille, pour leur soutien au quotidien et leurs mots de réconfort. Vous suivez chacun de mes choix. Les mots me manquent pour exprimer toute ma reconnaissance. Merci à toi Stéphane, mon grand Amour, pour ta patience et ta force qui me portent. A deux, nous sommes plus forts que tout ! Sommaire Sommaire Liste des abréviations i Liste des figures ii Liste des photos iii Liste des tableaux iiii Introduction générale – Objectifs 1 Partie 1 : Synthèse bibliographique I. Les champignons mycorhiziens à arbuscules (CMA) 6 I. 1. Taxonomie des champignons mycorhiziens arbusculaires 6 I. 2. Cycle de développement des champignons mycorhiziens arbusculaires 8 I. 2. 1. Phases asymbiotique et pré-symbiotique 9 II.2. 2. Phase symbiotique 11 I. 3. Bénéfices de la symbiose mycorhizienne à arbuscules 14 I. 3. 1. Bénéfices pour le CMA 14 I. 3. 2. Bénéfices trophiques pour la plante 15 I. 3. 3. Autres bénéfices 17 II. Mécanismes de tolérance chez les CMA (Revue 1) 19 III. Intérêt d’utiliser les CMA dans la phytoremédiation des sols contaminés par 46 les POP (Revue 2) Partie 2 : Matériels et méthodes I. Matériels biologiques 80 I.1. Matériel végétal 80 I.1. 1. Matériel végétal utilisé pour l’expérience in vitro 80 I.1. 2. Matériel végétal utilisé pour l’expérience en microcosme 80 I.2. Matériel fongique 81 II. Le sol historiquement pollué (site de l’Union) 82 II.1. Site de prélèvement 82 II.2. Caractérisation pédo-agronomique du sol 82 Sommaire II.3. Isolement et identification des CMA endogènes du sol 83 II.4. Détermination du potentiel mycorhizogène du sol 83 III. Dispositifs expériementaux 83 III.1. Cultures in vitro 83 III.1. 1. Milieu de culture 83 III.1. 2. Introduction du benzo[a]pyrène (B[a]P) dans le milieu de culture 84 III.1. 3. Cultures monoxéniques 85 III.2. Microcosmes 86 IV. Mesures des développements racinaires et fongiques 86 IV.1. Mesure du développement racinaire 86 IV.2. Mesure du développement fongique 87 V. Extraction et quantification des hydrocarbures dans le sol et le matériel 88 végétal V.1. Extraction des hydrocarbures (HAP totaux et biodisponibles et alcanes 88 totaux) V.2. Analyse des hydrocarbures (HAP et alcanes) 89 VI. Extraction et analyse des acides gras liés aux phospholipides (AGPL) à partir 91 du sol historiquement pollué VI.1. Extraction 91 VI.2. Trans-estérification 91 VI.3. Analyse des acides gras liés aux phospholipides 92 VII. Détection du peroxyde d’hydrogène (H2O2) 93 VIII. Analyse quantitative de l’expression de gènes dans les racines de luzerne et 93 les structures extraracinaires du CMA VIII.1. Extraction de l’ARN 93 VIII.2. Transcription inverse 94 VIII.3. PCR quantitative en temps réel (qPCR) 95 IX. Activités enzymatiques et dosage protéique 97 IX.1. Extraction du contenu protéique des racines et du CMA 97 IX.2. Dosage de l’activité peroxydase (POX) 97 IX.3. Dosage de l’activité superoxyde dismutase (SOD) 98 IX.4. Dosage de l’activité glutathion-S-trasnférase (GST) 98 IX.5. Dosage des protéines 98 Sommaire X. Analyses statistiques 99 Partie 3 : Résultats I. Publication 1: The persistent organic pollutant, benzo[a]pyrene, disturbs Medicago truncatula and Rhizophagus irregularis gene expressions Présentation de la publication 1 100 Sélection de couples d’amorces pour étudier l’expression de gènes 103 potentiellement impliqués dans la tolérance aux HAP chez les partenaires symbiotiques M. truncatula/ R. irregularis 1. Choix des gènes et des couples d’amorces pour M. truncatula 103 2. Choix des gènes et des couples d’amorces pour le CMA R. irregularis 103 3. Test des couples d’amorces de M. truncatula 106 4. Test des couples d’amorces de R. irregularis 109 Publication 1 113 II. Publication 2: Arbuscular mycorrhizal wheat inoculation promotes alkane and polycyclic aromatic hydrocarbon biodegradation : microcosm experiment on aged-contaminated soil Présentation de la publication 2 135 Publication 2 138 Partie 4 : Synthèse et perspectives I. Rôle de la mycorhization arbusculaire dans la protection des racines de M. 165 truncatula contre la toxicité d’un HAP lourd, le B[a]P I.1. Réponses au niveau transcriptionnel (géniques et enzymatiques) de la 165 symbiose M. truncatula/ R. irregularis et de chacun des partenaires après exposition au B[a]P I.2. Accumulation d’H2O2 et de B[a]P chez les partenaires symbiotiques M. 166 truncatula/ R. irregularis après exposition au B[a]P II.
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