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Association of Arbuscular Mycorrhizal Fungi and Crotalaria Spectabilis to Control the Burrowing Nematodes (Radopholus Similis) in Banana

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Association of Arbuscular Mycorrhizal Fungi and Crotalaria Spectabilis to Control the Burrowing Nematodes (Radopholus Similis) in Banana UNIVERSITE CATHOLIQUE DE LOUVAIN ****************** FACULTE DES BIOINGENIEURS ******************* EARTH AND LIFE INSTITUTE POLE OF APPLIED MICROBIOLOGY LABORATORY OF MYCOLOGY Association of arbuscular mycorrhizal fungi and Crotalaria spectabilis to control the burrowing nematodes (Radopholus similis) in banana Thèse réalisée par Mlle Abla ANENE en vue de l’obtention du grade de Docteur en Sciences agronomiques et ingénierie biologique Promoteur: Prof. Stéphane declerck Jury members: - Prof. Jacques Mahillon: President (UCL, Belgium) - Prof. Xavier Draye (UCL, Belgium) - Prof. Nicole Viaene (UGent/Belgium) - Prof. Annemie Elsen (UGent/Belgium) - Dr. Kodjo Tomekpe (CIRAD/Guadeloupe-France) Louvain-la-Neuve, Mars 2018 Acknowledgment En première position, j’adresse mes reconnaissances au Seigneur Dieu Tout Puissant de m’avoir accordé sa grâce et son amour tout au long de ce parcours si périlleux. Plusieurs personnes ont contribué de près ou de loin à la réalisation de cette thèse. Ainsi, je tiens à exprimer toute ma gratitude : A mon promoteur, Professeur Stéphane DECLERCK, pour m’avoir accueillie dans son laboratoire. En 2012 quand nous n’avions pas eu l’attribution de bourse de thèse de doctorat, aucune idée d’imaginer que le futur allait être si dur. Cependant tu as été toujours confiant qu’on arriverait à bout et tu n’as jamais douté de moi. Voilà on y est !!! Merci pour tout. A tout le personnel de CESAMM et de GINCO merci pour tous les moments de partage durant ces 7 longues années. Mes sincères remerciemments à Alain GUILLET de la Plate-forme technologique de Support en Méthodologie et Calcul Statistique (SMCS)/UCL pour tous vos assistances en analyses stat du début jusqu’à la fin de cette thèse. Mes remerciements aux membres du jury pour leurs contributions à l’amélioration du document final. A Kodjo TOMEKPE, merci du fond du cœur pour avoir guidé mes premiers pas dans la recherche et pour m’avoir soutenu du début jusqu’à la fin de cette thèse. Que Dieu veille sur ta santé et te confère une longevité afin que tu continues de transmettre ton savoir à la relève. A Mme Christine VOS, Nele SCHOUTEDEN et Mme Lut OOMS de la KUL, pour leurs encadrements et leurs soutiens inconditionnels au début de la thèse. Que Dieu vous bénisse. Mes remerciements à WAAPP-Togo (West Africa Agricultural Productivity Program), à la Direction et aux collègues de l’ITRA (Institut Togolais de Recherche Agronomique) pour leur soutien. 2 A l’asbl LEA (Les Enfants d’Aleth), les mots me manquent pour vous exprimer ma gratitude pour m’avoir soutenu tout au long de cette épreuve. Que Dieu vous bénisse. Particulièrement à la famille Pasteur MUNDEKE et à tous les membres de l’Eglise Cité de la Restauration et à Didier TOMEDONOU, vous m’aviez aidé à me sentir en famille et vous aviez été présent et à me motiver à chaque moment où je m’effondrais, sans oublier vos multiples aides de tous genres. Un seul mot : MERCI et que Dieu vous inonde de sa bénédiction. A toi Fernand LHOTELLERIE, un grand merci pour avoir été là pour moi à un moment crucial de ma thèse. Merci à tous mes amis et particulièrement la « famille des Prêtes» de LLN. Merci à tous mes frères et sœurs, pour vos encouragements et pour avoir pris soin de notre père et mère durant les longues périodes de leur maladie en mon absence. 3 Lists of illustrations FIGURE 1: RESEARCH OUTLINE OF THE THESIS ...................................................................... 19 FIGURE 2: CLASSIFICATION OF BANANA (GRIN, 2009) ......................................................... 27 FIGURE 3: BANANA PLANT (ANENE, 2016) .............................................................................. 28 FIGURE 4: CLASSIFICATION OF R. SIMILIS (COBB, 1893; THORNE, 1949) ............................ 30 FIGURE 5: SCHEMA OF R. SIMILIS (FROM ESSER ET AL., 1984) ............................................. 31 FIGURE 6: LIFE CYCLE OF R. SIMILIS (BROOKS, 2008) .......................................................... 33 FIGURE 7 : ARBUSCULAR MYCORRHIZAL FUNGI CLASSIFICATION (REDECKER ET AL., 2013). .......................................................................................................................................... 40 FIGURE 8: LIFE CYCLE OF AN AMF AND THE DIFFERENT STEPS DURING AMF DEVELOPMENT (GARG AND SHANDEL, 2010) ................................................................. 42 FIGURE 9: OVERVIEW OF THE POSSIBLE MECHANISMS BY WHICH ARBUSCULAR MYCORRHIZAL FUNGI CAN EXERT BIOCONTROL AGAINST PLANT-PARASITIC NEMATODES (SCHOUTEDEN ET AL., 2015). ..................................................................... 48 FIGURE 10: PLANT (A) OF CROTALARIA SPECTABILIS AND ITS FLOWERS (B) (MADDOX ET AL., 2009) AND SEEDS (C) (ANENE, 2006) ........................................................................ 50 FIGURE 11 BANANA SHOOTS UNDER IN VITRO CULTURE CONDITIONS (PROLIFERATING MEDIUM) ........................................................................................................................... 60 FIGURE 12 : MYCELIUM DONOR PLANT (MDP) IN VITRO CULTURE SYSTEM FOR THE PRODUCTION OF AMF. RC: ROOT COMPARTMENT; HC: HYPHAL COMPARTMENT. .... 65 FIGURE 13 : BANANA PLANTLETS IN THE HC OF THE MDP IN VITRO CULTURE SYSTEM FOR THE FAST AND HOMOGENOUS MYCORRHIZATION ........................................................... 66 FIGURE 14 : MYCORRHIZAL BANANA PLANTLET IN INDIVIDUAL SYSTEM (H-AMP IN VITRO CULTURE SYSTEM) ........................................................................................................... 67 4 FIGURE 15 : AMF-COLONIZED C. SPECTABILIS PLANTLETS IN THE H-AMP IN VITRO HYDROPONIC CULTURE SYSTEM ...................................................................................... 68 FIGURE 16: MICROCOSM POT DESIGN .................................................................................... 69 FIGURE 17: MICROCOSM SYSTEM SET WITH BANANA AND C. SPECTABILIS. THE TWO PLANTS ARE SEPARATED ABOVE-GROUND WITH FIXED TALPA NET ............................................. 70 FIGURE 18: ROOTS EXUDATES COLLECTION PROCESS ........................................................... 71 FIGURE 19 : A DIAGRAM TO SHOW HOW A MAGNIFIED INTERSECTION PERPENDICULAR TO THE LONG AXIS OF THE ROOT CAN BE MADE WHEN THE ROOT IS ALIGNED WITH ITS LONG AXIS AT AN ANGLE TO THE VERTICAL CROSSHAIR. ............................................... 72 FIGURE 20: ROOTS OF INOCULATED BANANA ......................................................................... 74 FIGURE 21: EXPERIMENTAL DESIGN TO MONITOR THE CHEMOTAXIS OF R. SIMILIS IN PRESENCE OF VARIOUS COMBINATIONS OF ROOT EXUDATES OF AMF-COLONIZED OR NON-COLONIZED C. SPECTABILIS OR BANANA PLANTLETS. .......................................... 121 FIGURE 22: IN VITRO HYDROPONIC CULTIVATION SYSTEM WITH AMF-COLONIZED C. SPECTABILIS PLANTLETS. ............................................................................................... 123 FIGURE 23: IN VITRO EVALUATION OF THE ORIENTATION OF NEMATODES AS A FUNCTION OF THE EXUDATES PRESENT IN THE CULTURE MEDIUM. .................................................... 124 FIGURE 24: IN VITRO CHEMOTAXIS BIO-ASSAY AND VISUALIZATION OF NEMATODE MOVEMENT ON THE MEDIUM. ........................................................................................ 125 FIGURE 25: PERCENTAGE OF IMMOBILE NEMATODES FOLLOWING INCUBATION IN DIFFERENT CONCENTRATIONS OF EXUDATES FROM AMF-COLONIZED AND NON- COLONIZED ROOTS OF C. SPECTABILIS. ......................................................................... 127 5 List of tables TABLE 1: NEMATODE POPULATION DENSITIES IN THE ROOTS OF MYCORRHIZAL (+AMF) AND NON-MYCORRHIZAL (–AMF) MUSA ACUMINATA CV. YANGAMBI KM5 PLANTLETS GROWN UNDER IN VITRO CULTURE CONDITIONS, SIX WEEKS AFTER INOCULATION WITH 100 ASEPTICALLY PRODUCED JUVENILES AND ADULTS OF R. SIMILIS (NEM) ................ 84 TABLE 2 : OBSERVED DATA OF ROOT FRESH WEIGHT (RFW), AND SHOOT DRY WEIGHT (SDW) OF MYCORRHIZAL BANANA PLANTLETS GROWN IN PRESENCE/ABSENCE OF C. SPECTABILIS AND INFESTED OR NOT WITH R. SIMILIS (+M+C+N, +M+C-N, +M-C+N, +M-C-N) AND NON-MYCORRHIZAL BANANA PLANTLETS GROWN IN PRESENCE/ABSENCE OF C. SPECTABILIS AND INFESTED OR NOT WITH R. SIMILIS (-M+C+N, -M+C-N, -M- C+N, -M-C-N).. ............................................................................................................. 100 TABLE 3: OBSERVED DATA OF ROOT FRESH WEIGHT (RFW) AND SHOOT DRY WEIGHT (SDW) AND NUMBER OF NODULES ON ROOTS OF C. SPECTABILIS PLANTS GROWN IN PRESENCE/ABSENCE OF AMF AND R. SIMILIS. (+M+C+N, +M+C-N, -M+C+N, -M+C- N). ................................................................................................................................... 101 TABLE 4: OBSERVED DATA OF PERCENTAGE OF ARBUSCULES, SPORES/VESICLES AND TOTAL COLONIZATION BY AMF IN MYCORRHIZAL BANANA PLANTS GROWN IN PRESENCE/ABSENCE OF C. SPECTABILIS AND INFESTED OR NOT WITH R. SIMILIS (+M- C+N, +M+C+N, +M+C-N, +M-C-N) ........................................................................... 102 TABLE 5: OBSERVED DATA OF PERCENTAGE OF ARBUSCULES, SPORES/VESICLES AND TOTAL COLONIZATION BY AMF IN ROOTS OF C. SPECTABILIS IN POTS OF MYCORRHIZAL BANANA PLANTS GROWN IN PRESENCE/ABSENCE OF R. SIMILIS (+M+C+N, +M+C-N). ........................................................................................................................................ 103 TABLE 6: NEMATODE POPULATION DENSITIES, MULTIPLICATION RATE AND ROOT NECROSIS INDEX (RNI) IN THE ROOTS OF MYCORRHIZAL BANANA PLANTS GROWN IN PRESENCE/ABSENCE OF C. SPECTABILIS (+M+C+N,
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