MEMOIRE DE FIN D'etudes Evaluation De L'activité Antagoniste

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MEMOIRE DE FIN D'etudes Evaluation De L'activité Antagoniste REPUBLIQUE ALGERIENNE DEMOCRATIQUE ET POPULAIRE MINISTERE DE L’ENSEIGNEMENT SUPERIEUR ET DE LA RECHERCHE SCIENTIFIQUE UNIVERSITE BLIDA 1 FACULTE DES SCIENCES DE LA NATURE ET DE LA VIE DEPARTEMENT DES BIOTECHNOLOGIES MEMOIRE DE FIN D’ETUDES En vue de l’obtention du diplôme de Master 2 Option : Biologie des Interactions Plantes-Microorganismes Présenté par : BOUKERCHAOUI Saliha Evaluation de l’activité antagoniste des filtrats de cultures d’un isolat de Trichoderma sp. Vis- à-vis de quelques champignons phytopathogénes Soutenu devant le jury : BENCHABANE M. Professeur U. Blida 1 Président AMMAD F. M.C.B. U. Blida 1 Promotrice BOUCHENAK F. M.C.B. U. Blida 1 Examinatrice YALA A. Doctorante U. Blida 1 Invitée ANNEE UNIVERSITAIRE 2016/2017 Remerciements Ce travail a été réalisé au niveau du laboratoire de mycologie du département des Biotechnologies de l’Université de Blida1. Et au laboratoire de mycologie à l’institut national de la protection des végétaux d’El Harrach (INPV). Je remercie, en premier lieu, ALLAH le tout puissant de m’avoir donné le courage, la force et la volonté pour bien mener ce travail. Au terme de ce travail, Je tiens à exprimer toute ma gratitude, ma reconnaissance et mes sincères remerciements à ma promotrice Mme Ammad F. d’avoir accepté de m’encadrer, diriger et donné la chance de travailler ce sujet, sa confiance, sa disponibilité et la générosité qu’elle a m’ont accordée pour faire avancer ce travail. Je remercie sincèrement Pr. BENCHABANE M de m’avoir honoré en acceptant de présider le jury de mon travail. Je remercie également à Mlle BOUCHENAK f. et Mlle YALA A. d’avoir accordé leurs temps pour honneur d’évaluer et d’enrichir ce travail. Je remercie très vivement, Mme C. Zouai, Mlle Ousaaid Yamina de service mycologie /INPV de m’avoir permis de réaliser une partie de mon travail, la préparation des extraits de filtrat de culture et m’a aidé à réaliser l’extraction des métabolites secondaires au niveau de laboratoire des pesticides. Mes remerciements vont aussi à, Mlle khalida, Mlle Hadjer et a toutes les personnes de l’INPV pour leurs aides et disponibilités pour répondre à mes questions durant cette recherche. A Mme AIT SAIDI N., de m’avoir fait profiter des connaissances et pour le soutien et conseils qu’elle m’a accordé concernant l’analyse enzymatique. Un grand merci également à FADIL Djamila, ingénieur du laboratoire, pour sa disponibilité et ses encouragements durant toute l’expérimentation. Je remercier très spécialement Mr Izri Hamza, qui a été toujours là pour moi, pour sa grande part dans la réalisation de cette recherche et pour ces conseils sérieux, Mercie pour votre amitié. Je tiens a remercié Mr Souaber Mohamed, pour son aide et son support moral et encouragement tout au long de ma démarche. J’adresse mes plus sincères remerciements a tout ma famille pour leurs encouragements et surtout Mohamed Reda, Ikram et saada. Enfin, que tous ceux, de près ou de loin, dont l’assistance ou les conseils m’ont permis de mener à bien ce travail, trouvent ici l’expression de ma profonde gratitude et de mes salutations les plus respectueuses. Dédicaces À cœur vaillant rien d’impossible À conscience tranquille tout est accessible Quand il y a la soif d’apprendre Tout devient facile pour arriver à nos fins Malgré les obstacles qui s’opposent En dépit des difficultés qui s’interposent Les études sont avant tout Notre unique et seul atout Ils représentent la lumière de notre existence Je dédie ce modeste travail à tous ceux qui me sont chers : A mon père que nulle dédicace ne puisse exprimer mon respect, mon amour éternel et ma considération pour l’encouragement contenu les sacrifices que vous avez consenti pour mon instruction et mon bien être. Je vous remercie pour tout le soutien et l’amour que vous me portez depuis mon enfance et j’espère que votre bénédiction m’accompagne toujours. Que ce modeste travail soit l’exaucement de vos vœux tant formulés, le fruit de vos innombrables sacrifices. Puisse Dieu, vous accorder santé, bonheur et longue vie. A ma très chère Maman, et ma deuxième mère Saada, merci pour ton amour, ton inquiet, tes encouragements et ton soutien durant toute ma vie, je t’aime énormément. À mes très chères sœurs Fatima El zahraa, Khoulod, Ikram et més Cher frères Riadh et Yahia, à qui je dois tout l’amour, avec tous mes vœux de les voir réussir dans leurs vies. A mes amies, mes collègues de la promotion, à qui je souhaite le succès, pour l’amitié qui nous a toujours unis, qui sans leur encouragement ce travail n’aura jamais vu le jour. A tous ceux qui m’ont encouragé, soutenus et supporté pour que ce travail puisse s’accomplir Liste des abréviations BDA Black Dead Arm . °C Degré Celsius. µl Microlitre. AIA Aide indole acétique. BD Botriyosphaeria dothidea FNRDA Fond National de régulation et développement agricole). FTIR Fourier Transformed Infra Red spectroscopy g gramme. GC-MS Chromatographie en phase gazeuse liée à la spectrométrie en mass . GLM Generalized –linear Model. ml Millilitre . O.I.V Organisation international de vine. P.N.D.A. Le plan national de développement Agricole PDA Potato Dextrose Agar. PDB Potato Dextrose Broth. PGPF Plant Growth Promoting Fungi. rpm rotation par minute. Tsp Trichoderma sp. Listes des Annexes Annexe 1 Milieux de culture utilisés 110 Annexe 2 Spectres obtenues par l’analyse FTIR de Trichoderma sp 112 Annexe 3 les moyennes des nécroses calculer sur les variétés testées en cm 50 Liste des Figures Figure 1 Les symptômes au niveau du bois de Vitis vinifera atteintes 7 d’eutypiose, (a) et (b) rameaux rabougris. (c) nécrose brune en forme de V (Bertsch et al., 2013). Figure 2 Les symptômes au niveau du bois et des feuilles de Vitis vinifera 8 atteintes d’esca (Bertsch et al., 2013). Figure 3 Les symptômes au niveau du bois et des feuilles de Vitis vinifera de 11 Black Dead Arm également appelé chancre à Botryosphaeria, Figure 4 Les cinq sections de Trichoderma reconnus par Bissett (1991). 19 Figure 5 Les mécanismes d’induction de la résistance systémique par les 24 souches de Trichoderma (Irina et al., 2011). Figure 6 Mécanisme de mycoparasitisme des Trichoderma (Druzhinina et al., 26 2011) Figure 7 Schéma montrant la confrontation à équidistance du l’agent pathogène 33 et de Trichoderma sp.sur milieu PDA Figure 8 Schéma montrant la confrontation à distance 34 Figure 9 Méthodologie de l’inoculation des mycéliums pathogène ou de 36 pathogène et l’antagonistes. Figure 10 Ré-isolement des souches pathogènes sur milieu PDA. 38 Figure 11 Aspect macroscopique et microscopique de Trichoderma sp, 40 Figure 12 Aspect macroscopique et microscopique des Botryosphaeriaceae. 41 Figure 13 Production de la chitinase par Trichoderma sp. 43 Figure 14 Production d’endoglucanase après 7 jours d’incubation à 28°C. 44 Figure 15 Production des amylases par Trichoderma après 3 jours d’incubation à 44 28°C . Figure 16 Formation d’un halo brun-foncé autour des colonies de Trichoderma 45 caractéristique de la production de ligninase après 2 jours d’incubation. Figure 17 Production de la lipase traduite par la présence d'un halo blanc opaque 45 autour de colonie de Trichoderma. Figure 18 Apparition d’un Halo transparent autour de la colonie de Trichoderma 46 sp. Figure 19 Production d’AIA sur le milieu liquide additionné de tryptophane. 46 Figure 20 Pourcentage d’inhibition de la croissance mycélienne des 53 Botryosphaeria par Trichoderma sp après six jours d’incubation à 25 °C Figure 21 Inhibition de la croissance mycélienne des Botryosphearia (B), en 54 comparaison avec leur témoin(A), par confrontation directe avec Trichoderma sp. Après 6 jours d’incubation à 25°C. Figure 22 Action mycoparasitaire de l’isolat Trichoderma sp montrant une 55 surcroissance et une sporulation abondante sur la colonie de pathogène. Figure 23 Sporulation de Trichoderma sp en masse sur les hyphes de 55 Botryosphaeria dothidea (x40) Figure 24 Apparition de grandes vacuoles à l’intérieur du mycélium de pathogène 56 (x40) Figure 25 la lyse Présence de petites vésicules de Botryosphaeria sp2 (x40) 56 Figure 26 Pourcentage d’inhibition de la croissance mycélienne de 57 Botryosphaeria par Trichoderma sp après six jours d’incubation à 25 °C Figure 27 Pourcentage d’inhibition de mycélium des pathogènes par le filtrat de 58 culture de Trichoderma après 5jours d’incubation Figure 28 Pouvoir inhibiteur de filtrat de culture de Trichoderma sp sur le 59 mycélium des pathogènes après 5 jours d’incubation à 25 °C. Figure 29 Des nécroses internes et des chancres sur la variété Datale 68 Figure 30 Evolution des tailles des nécroses des boutures inoculées par le 68 pathogène Figure 31 Evolution des tailles des nécroses des boutures inoculées par le 69 pathogène et traités par Trichoderma sp. Figure 32 Les nécroses présentées sur les boutures de Vitis vinifera (variété 70 Victoria). (A, C, E, G) : inoculé par Botryosphaeria dothidea (B, D, F, H) : co-inoculation de Trichoderma sp et Botryosphaeria dothidea au bout de 7jours (A, B), 14jours (C, D), 21jours (E, F) et 28jours (G, H). Figure 33 Les nécroses présentées sur les boutures de Vitis vinifera (variété Red 70 glob). (A, C, E, G) : inoculé par Botryosphaeria dothidea (B, D, F, H) : co-inoculation de Trichoderma sp et Botryosphaeria dothidea au bout de 7jours (A, B), 14jours (C, D), 21jours (E, F) et 28jours (G, H). Figure 34 Les nécroses présentées sur les boutures de Vitis vinifera (variété 71 Muscat italia). (A, C, E, G) : inoculé par Botryosphaeria dothidea (B, D, F, H) : co-inoculation de Trichoderma sp et Botryosphaeria dothidea au bout de 7jours (A, B), 14jours (C, D), 21jours (E, F) et 28jours (G, H).
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