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A Page De Garde Model Vrpg Juil Département de Biotechnologie Thèse de Doctorat en Microbiologie Thème Diversité taxonomique et symbiotique des rhizobia associés aux Acacia sp. d’Algérie Présentée par : Boukhatem Zineb Faiza Dirigée par : Mr BEKKI Abdelkader , Professeur à l’Université d’Oran. Devant les membres de jury constitué de: Président e : FYAD Zohra , Professeur à l’Université d’Oran. Examinateur : HADJAJ AOUEL Seghir, Professeur à l’Université d’Oran. Examinateur : ABDELWAHED Djaml Eddine, Professeur à l’Université de Tlemcen. Examinateur : de LAJUDIE Philippe, Directeur de recherches à l’IRD, France. Co-Directeur de thèse : GALIANA Antoine , Directeur de recherches au CIRAD, France. Dédicace Je dédie ce travail à ma mère, ses sacrifices ont pavé mon chemin vers la réussite, je ne saurais lui exprimer ma gratitude pour tout ce qu’elle a inculqué en moi. J’espère que ce modeste travail récompensera en partie son dévouement. A feu mon père qui aura toujours une place dans mon cœur. A mes trois trésors, mon mari ma fille et mon fils qui donnent un sens à ma vie. A mon frère, mes deux sœurs ainsi que les prunelles de mes yeux, mes petites nièces chéries. A toute ma famille, ma belle famille et mes amis. RemerciementRemerciementssss Merci tout d’abord à Dieu miséricordieux de m’avoir donné la force d’accomplir cette tâche. Ce travail s’est déroulé en alternance au LBRAP (Laboratoire de Biotechnologie des Rhizobiums et amélioration des plantes à l’université d’Oran, Es-Senia et au LSTM (Laboratoire des Symbioses Tropicales et Méditerranéennes) de Montpellier, France. Je remercie Pr. BEKKI Abdelkader d’avoir accepté d’encadrer ce travail et pour tous ses conseils avisés. Un grand merci au Dr. GALIANA Antoine pour tous les efforts fournis, pour ses approches intéressantes et pour sa disponibilité qui lui a gâché bien des weekends et des vacances. Ainsi qu’aux directeurs successifs du LSTM (Laboratoire des Symbioses Tropicales et Méditerranéennes) de Montpellier : DREYFUS Bernard et LEBRUN Michel pour avoir accepté ma candidature à des stages plus que fructueux qui m’ont permis d’avancer dans mon travail. Je tiens à remercier les membres du jury : Pr. FYAD qui a accepté sans hésitation de présider ce jury, Pr. HADJAJ et Pr.ABDELWAHED qui m’ont accordé leur temps afin d’examiner ce travail en cette fin d’année toujours surchargée, et enfin un grand merci au Pr. de LAJUDIE qui répond toujours présent quant on a besoin de lui. Je ne saurais remercier ma collègue et amie Dr MERABET Chahinez pour son aide précieuse sur la construction phylogénétique, ainsi que pour son soutient et sa bonne humeur contagieuse. J’exprime aussi ma reconnaissance à tous mes collègues du département de biotechnologie, enseignants, personnel technique et à l’équipe du laboratoire de Laboratoire de Biotechnologie et Amélioration des Plantes, enseignants et post-graduants pour leurs encouragements. Je ne saurais oublier de remercier toute l’équipe du LSTM (Laboratoire des Symbioses Tropicales et Méditerranéennes) de Montpellier ; Odile pour son aide précieuse, Christine pour son humanisme, Philippe et tous les autres : chercheurs et doctorants qui m’ont aidé ne serait ce que d’un mot, d’un sourire. Mes courts séjours m’ont beaucoup enrichi autant sur le plan scientifique qu’humain. Mes remerciements s’adressent aussi à la direction des forêts de toutes les wilayas que j’ai visitées, les moyens généraux ainsi que l’INRF spécialement Salah, Mme Sahki ainsi que Réda et Ibrahim, nos guides à Tamanrasset. Enfin, j’exprime toute ma gratitude à toute ma famille, mes amis d’ici et d’ailleurs que j’ai délaissés mais qui ont été compréhensifs et patients et à toutes les personnes qui m’ont encouragées, soutenues durant les moments difficiles. Une thèse est un investissement en temps, en énergie, en santé, avec quelques manips qui ne vont pas dans le sens qu’on aurait voulu, mais avant tout, c’est une grande expérience humaine, alors MERCI à tous ceux qui y ont contribué. Sommaire Sommaire Chapitre I : Recherche bibliographique I : « Les rhizobia » I-1- Définition des rhizobia…………………………………………….……………. 01 I-2- La symbiose légumineuse-rhizobium : définition et évolution…..…………… 02 I-3- Formation des nodules……...…………………………………………………… 03 I-4- Taxonomie des BNL……...……………………………………………………... 05 I-4-1- Outils de la taxonomie bactérienne………………………………...…………. 05 I-4-2- Position taxonomique actuelle des BNL…..………………………..………… 09 I-4-3- Taxonomie des rhizobia associés aux Acacias………………………..……... 12 I-5- Spectre d’hôte des rhizobia associés aux Acacias…………………………….… 19 I-6- Effet des contraintes édaphiques sur la croissance des rhizobia…………….….. 19 II: « Les Acacias » II-1- Généralités……………………………………………………………………… 25 II-2- Origine et évolution de la nodulation chez les légumineuses………………….. 25 II-3- Taxonomie des Acacia …………………………………………………………. 26 II-4- Répartition des Acacias dans le monde………………………………………… 29 II-5- Intérêts et importance des Acacias……………………………………………... 29 II-6- Les Acacias en Algérie…………………………………………………………. 34 II-6-1- Description et répartition géographique des Acacias étudiés………………... 35 II-6-2- Utilisation et caractéristiques des Acacias étudiés…………………………... 40 II-7- Résistance des Acacias aux contraintes édaphiques…………………………… 42 III: « La fixation d’azote atmosphérique » III-1- Introduction……………………………………………………………………. 46 III-2- Comparaison entre les arbres et les plantes annuelles fixateurs d’azote ……... 46 III-3- Méthodes d’estimation de la fixation d’azote atmosphérique………………… 47 III-3-1- Méthodes indirectes……………………………………………………….. 47 III-3-2- Méthodes directes (isotopiques)………………………………………… 49 III-4- Le potentiel fixateur de N 2 des Acacias ………………………………………. 54 III-5- Classification des Arbres Fixateurs d’A zote selon leur potentiel de fixation du N2……………………………………………………………………………………………………………………………… 57 Chapitre II : Etude expérimentale Partie I : Caractérisation des rhizobia associés aux espèces d’ Acacia d’Algérie Matériel et méthodes: Caractérisation des rhizobia Introduction…………………………………………………………………………. 58 Méthodologie………………………………………………………………………… 58 1-Echantillonnage………………………………………………………………….. 58 2-Analyse physico-chimique du sol……………………………………………….. 61 3- Isolement des rhizobia………………………………………………………….. 61 3-1-Piégeage …………………………………………………………………. 61 3-2- Isolement et purification des souches ………………………………….. 63 4- Test de nodulation et d’efficience ……………………………………………… 64 5- Analyse statistique ……………………………………………………………… 66 Sommaire 6- Caractérisation phénotypique des isolats……………………………………….. 67 6-1- Tolérance à la température. ……………………………………………. 67 6-2- Tolérance aux pH……………………………………………………….. 67 6-3- Tolérance à la salinité…………………………………………………… 67 7- Phénodendogramme……………………………………………………………. 67 8- Caractérisation génétique des isolats……………………………………………. 69 Résultats et discussion : Caractérisation des rhizobia 1- Distribution des Acacias…………………………………………………..... 73 2- Caractérisation des sites et sols prospectés …………………………………. 75 3- Taux de germination des graines après les différentes méthodes de désinfection et de scarification………………………………………………. 77 4- Impact de la profondeur d’échantillonnage dans les lits d’oueds desséchés sur l’isolement des populations rhizobiennes………………………………... 79 5- Isolement, purification et statut symbiotique des souches …………………. 82 6- Caractérisation génétique par le séquençage partiel du 16S rARN…………. 88 7- Détermination de la spécificité d’hôte des BNL isolées par AFC ……........ 98 8- Tolérance des souches à la salinité, température et pH……………………… 100 9- Tests d’efficience des souches 108 d’Acacias…………………………………….. 10- Caractérisation phénotypique des souches isolées d’Acacias phénodendogramme 115 Partie II : Potentiel fixateur d’azote des Acacia en Algérie Matériel et méthodes : Potentiel fixateur d’azote des Acacia en Algérie Introduction…………………………………………………………………………. 121 1- Méthode de la dilution isotopique par enrichissement du sol en 15 N …………… 122 a- Protocole d’échantillonnage sur le terrain ……………………………................ 122 a-1 Description des sites de prélèvement …………………………………….. 122 a-2 Prélèvement du sol ………………………………………………………… 123 b- Protocole expérimental en serre……………………………………… ………… 123 c- Analyse du sol………………………………………………………………….. 127 2- Abondance naturelle en 15 N………………………………………………………. 127 Résultats et discussion : Potentiel fixateur d’azote des Acacia en Algérie 1- Résultats des analyses du sol des trois sites étudiés…………………………….. 130 2- Effet du sol sur la croissance de trois espèces d’ Acacia …………………………… 131 3- Effet de l’inoculation sur la croissance de trois espèces d’ Acacia sur quatre types de sols………………………………………………………………………………..... 136 4- Estimation du potentiel fixateur de trois espèces d’ Acacia sur différents types de sols par la méthode de la dilution isotopique en 15 ………………………................ 140 5- Estimation du potentiel fixateur in situ de trois espèces d’ Acacia sur différents types de sols par la métho de de l’abondance naturelle………………………….......... 146 Conclusion et perspectives ………………………………………………………….. 152 Références bibliographiques Annexes Liste des tableaux Page Tableau 1 Classification des Bactéries Nodulant les Légumineuses (Weir, 10 2011). Tableau 2 Classification des espèces de rhizobia nodulant les Acacias. 13 Tableau 3 Les classifications les plus importantes dans le genre Acacia de 27 Bentham (1875) à Maslin et al . (2003) Tableau 4 Nom génériques et infragénériques de Acacia sens. lat . 28 Tableau 5 Classification d’Acacia sens. lat. dans le monde (Maslin et al., 29 2003). Tableau 6 Caractéristiques morphologiques, distribution géographique
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